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Metal stoichiometry, coenzyme binding, and zinc and cobalt exchange in highly purified yeast alcohol dehydrogenase
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
184, 505-517 (1977)
Metal Stoichiometry, Coenzyme Binding, and Zinc and Cobalt Exchange in Highly Purified Yeast Alcohol Dehydrogenasel ARTHUR J. SYTKOWSKI* Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts 02115 Received
May 27, 1977; revised
August
12, 1977
Yeast alcohol dehydrogenase, purified from baker’s yeast under conditions which exclude contamination by extraneous metal ions, is homogeneous by analytical ultracentrifugation and disc gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a molecular weight of 149,000 as determined by ultracentrifugation time-lapse photography and exhibits specific activities of 430 to 480 U/mg. Zinc analysis by three independent, highly sensitive methods, i.e., atomic absorption spectrometry, atomic fluorescence spectrometry, and microwave-induced plasma emission spectrometry, demonstrates 4 g-atom of catalytically essential Zn per mole of enzyme. No other metal atoms are present in stoichiometrically significant quantities as assessed by emission spectrography. The stoichiometry of coenzyme binding, 4 mol of NADH/mol of enzyme, is identical to that of zinc, consistent with one coenzyme binding site and one zinc atom per enzyme subunit. Conditions for exchange of the four catalytically essential zinc atoms with ““Zn have been developed. These atoms exchange identically under all conditions examined. The resultant radiolabeled enzyme, [(YADH)““Zn,], has the same metal content, specific enzymatic activity, and coenzyme binding properties as the native enzyme. The ““Zn of this enzyme serves to monitor the extent and site specificity of cobalt replacement. The fully cobalt-substituted enzyme, [(YADH)Co,], has a specific activity of 80 U/mg, 17% that of the Zn enzyme, and exhibits absorption and circular dichroic spectra which are consistent with coordination by one or more sulfur ligands in a distorted tetrahedral geometry.
Yeast alcohol dehydrogenase was first demonstrated to be a zinc metalloenzyme by Vallee and Hoch (11, who found a stoichiometry of 4.1 g-atom of catalytically essential zinc per mole of enzyme, employing both emission spectrographic and microchemical spectrophotometric methods for metal analysis. This stoichiometry is identical to that reported for NADH binding to the enzyme (2) and is consistent with one active site zinc atom and one coenzyme binding site per subunit.
Subsequently, Wallenfels and Sund (3, 4) and Sund (5) documented in great detail the ready acquisition of adventitious zinc by YADH.3 They showed that the enzyme could bind up to 35 g-atom of Zn per mole and that the excess Zn could be removed by various methods including recrystallization and dialysis against EDTA. Recently, Coleman and Weiner (6) isolated YADH from yeast grown in the presence of zinc or manganese and found 3.7 -r- 0.6 g-atom of either metal per mole of enzyme, a stoichiometry consistent with earlier re-
’ This work was supported by Grant-in-Aid GM15003 from the National Institutes of Health of the Department of Health, Education, and Welfare. 2 Special Fellow of the National Institute of Genera1 Medical Sciences, National Institutes of Health. Present address: Department of Pediatrics, Harvard Medical School, Children’s Hospital Medical Center, Division of Hematology-Oncology, 300 Longwood Avenue, Boston, Massachusetts 02115.
pOrtS.
3 Abbreviations used: YADH or [(YADH)Zn,], native yeast alcohol dehydrogenase; [ (YADH)““Zn,], radiolabeled yeast alcohol dehydrogenase; l(YADH)Co,], cobalt-substituted yeast alcohol dehydrogenase; NADH, nicotinamide adenine dinucleotide, reduced form; CD, circular dichroism; LADH, native horse liver alcohol dehydrogenase. 505
Copyright All rights
0 1977 by Academic Press, Inc. of remoduction in anv form reserved.
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ARTHUR
J. SYTKOWSKI
Preliminary studies in this laboratory with YADH from commercial sources revealed zinc stoichiometries in excess of 4 g-atom per mole and variable specific activities. A reassessment of coenzyme binding by Dickinson (7) resulted in an NADH/ YADH stoichiometry of 2:1, in contrast to 4:l (2, 8) or 51 (4) reported earlier. The elucidation of two classes of zinc atoms, catalytic and noncatalytic, in horse liver alcohol dehydrogenase (9-11) and partial sequence homologies between YADH and LADH (12) suggested to Branden et al. (13) that the yeast enzyme might contain 8 g-atom of Zn per mole. These considerations prompted a careful reinvestigation of the intrinsic zinc content of well-characterized YADH by three highly sensitive analytical methods (14). Despite the accumulated evidence that homogeneous enzyme contains only 4 g-atom of intrinsic Zn per mole (1, 3, 6, 14, 15), reports of higher metal contents continue to appear (16). The present study describes the physicochemical properties, specific enzymatic activity, metal content, and coenzyme binding characteristics of YADH prepared under rigorously controlled conditions. The results confirm that there are 4 gatom of catalytically essential Zn per mole of YADH and that the enzyme binds 4 mol of coenzyme. In addition, the native Zn atoms have been replaced first by 65Zn and subsequently by Co resulting in [(YADH)‘j5Zn,l and [ (YADH)CoJ, respectively. The cobalt-substituted enzyme is catalytically active and contains a sensitive spectral probe which permits the investigation of the intrinsic metal atoms by a variety of spectroscopic techniques. MATERIALS
AND
METHODS
Yeast. Fresh baker’s yeast was obtained from Federal Yeast Corp. as l-lb cakes, which were finely crumbled and spread in a uniform l-cm layer onto fiberglass window screens covered with two layers of grade 50 cotton cheesecloth. The screens were stacked horizontally with a X-cm space between them, and the yeast was allowed to air dry for 7 days at 22°C in an air-conditioned room with continuous air circulation. Dried yeast not used immediately was sealed in polyethylene bags and stored at
4°C for not longer than a few weeks (17). For comparative purposes, dried yeast was also obtained from P-L Biochemicals in two forms: “baker’s active-dried” and “compressed baker’s” YADH. Commercial preparations of enzyme were obtained either as a lyophilized powder or as a crystalline suspension from Boehringer-Mannheim Corp. and as lyophilized powders from Sigma Chemical Co. and Worthington Biochemical Corp. Reagents and purification. Phosphate buffers were prepared from analytical reagent grade Na2HP0, (Fisher). A 0.5 M stock solution was adjusted to pH 7.5 with HCl and adventitious metals were removed by solvent extraction with 0.01% dithizone in CCL (18). Other buffer concentrations and pH values were attained by appropriate dilution of this stock with metal-free water followed by pH adjustment. Sodium acetate buffer solutions were prepared similarly from a 1 M stock solution of ultrapure NaC2H,01 (anhydrous form) (Alfa Products, Ventron Corp.). Metal-free water was obtained by distillation followed by passage through two mixed-bed ion exchange resin cartridges (Cole-Palmer) in series and stored in a sealed polyethylene container. Air venting into the storage container on withdrawal was passed through a O.&pm filter to prevent airborne contamination. Water prepared and stored in this manner was found to be free of metals when analyzed by emission spectrography, microwave emission spectrometry, and atomic fluorescence spectrometry, down to levels of at least 10e4 pg/ml. Metal-free hydrochloric acid used for pH adjustment of buffers was prepared from metalfree water in cleaned (see below) polypropylene containers by isothermal distillation (19). Metal ion solutions for the exchange experiments were made from 1 M stock solutions of spectrographically pure ZnSO,. 7H,O or CoC12.6H10 (Johnson-Matthey Chemicals, Ltd.), which were analyzed for metal content. A saturated solution (25°C) of (NH&SO, (ultrapure, special enzyme grade, Schwarz/Mann) was prepared in metal-free water, and the pH was adjusted to 7.0 with isothermally distilled NH,OH. Sodium pyrophosphate buffer (0.1 M, pH 8.8) and 1 mM EDTA solutions (tetrasodium salt, pH 8.0) were made from analytical reagent grade materials. pNADH (grade III) and fi-NAD+ (grade III) were obtained from Sigma Chemical Co. Glassware. Whenever possible, plastic containers were employed and the use of glassware was avoided. Glassware was cleaned by soaking for several hours in a 1:l solution of HN03/H2S04, rinsing with metal-free water, soaking for 24 h in a 3% solution of Count-Off decontaminant/detergent (New England Nuclear), and then thoroughly rinsing with metal-free water. Plastic ware (polyethylene, polypropylene, polystyrene) was obtained new, rinsed in tap water to remove dust, soaked in 3%
PROPERTIES
OF YEAST
ALCOHOL
Count-Off for 24 h, and then thoroughly rinsed with metal-free water. After use, the plastic vessels were cleaned by repetition of the same procedure without exposure to highly reactive acids which damage the hydrophobicity of the surface. This procedure rendered these plastic materials completely free of any detectable metal comtamination. Dialysis tubing. Cellulose dialysis tubing (Vs-in. flat width) was prepared by heating cut lengths to 70°C in four changes of metal-free water. They were then transferred to a plastic container, rinsed in two changes of metal-free water, and stored at 4°C in metal-free water. All dialysis tubing, plastic closure clips, magnetic stirbars, etc., were handled with disposable polyethylene gloves (Cole-Parmer, Chicago) to prevent contamination. Enzyme purification. YADH was prepared from dried yeast according to the method of Hvidt and Kagi (17). Using fresh yeast, enzyme crystals having a silken sheen readily appeared at 25% saturation with ammonium sulfate. The specific enzymatic activity was 310-330 U/mg after two recrystallizations and 400-425 U/mg after four recrystallizations (see Results). Further purification was achieved by gel filtration through Sephadex G-200 (Pharmacia) in a 2.5 x 50-cm column, 0.24 ml/min ascending, 20 mM sodium phosphate, pH 7.5. Fractions (1.4 ml) having the highest uniform specific enzymatic activity were combined and concentrated by pressure dialysis (Diaflo PM-10 membrane, Amicon). Membranes were soaked for 24 h in several changes of metal-free water prior to use. Metal analysis. Three sensitive analytical systems for metal determinations were used in this investigation: atomic absorption spectrometry with total consumption, flame atomization and a longpath absorption tube (20), atomic fluorescence spectrometry with continuous sample introduction, and graphite furnace atomization (21, 221, and low-pressure, microwave-induced plasma emission spectrometry employing microvolume sampling (23). In addition, an emission spectrograph (Jarrell-Ash) served to determine the simultaneous multielement composition in ascertaining the presence and stoichiometries of metals other than zinc in YADH (24). All metal determinations reported are the averages of three to five measurements, and replicate measurements all fall within a range of 25%. Radioactivity measurements. The 6SZn radioisotope was measured by y emission spectrometry (Model 1185, Searle Analytic, Inc.). Sufficient counts were accumulated for each measurement to assure r2% reproducibility in the counting statistics at the 95% confidence level. BSZn (sp act = l-10 Ci/g) (New England Nuclear) was added to the buffers used for exchange experiments at a level of 0.1 mCi/lOO ml. Enzymatic actiuity. Activity was determined spec-
DEHYDROGENASE
507
trophotometrically by measuring the rate of formation of NADH at 340 nm and 25°C (1). The specific enzymatic activity reported herein is expressed in units (U) per milligram of protein, where 1 U = 1 +mol of NADH formed per minute. The concentrations of NADH and YADH were determined spectrophotometrically using extinction coefficients of A$: = 6.22 and&:‘,‘,” = 1.23, respectively (see Results). The 3.0-ml reaction mixture contained 5 Fmol of NAD+ and 1 mmol of ethanol in 16.7 mM pyrophosphate buffer, pH 8.8. The assay was initiated with 0.5-l pg of enzyme. Dry weight determinations. The dry weight (25) of protein solutions of known volume and the accurately measured absorbance at 280 nm were established for homogeneous YADH as follows. Samples were dialyzed at 4°C vs several changes of metalfree water, the pH of which had been adjusted to -8 with NH,OH. The absorbance at 280 nm was measured carefully and known volumes were transferred by calibrated pipet into constant-weight beakers, as were similar volumes of the final dialysis buffer for a blank determination. The samples were frozen, lyophilized for 24 h, and weighed on a semimicroanalytical balance (calibrated with N.B.S. Class M weights). They were then heated at 110°C for 1 h, cooled for 2 h, and weighed. This latter process was repeated over several cycles to verify that all the water had been removed by lyophilization. All containers were handled in accordance with standard gravimetric procedures to prevent any surface contamination which might alter their weights. Analytical ultracentrifugation. Sedimentation velocity experiments were carried out at 56,000 rpm at 21°C in a Spinco Model E ultracentrifuge (Beckman), at protein concentrations of 5-10 mg/ml in 20 mM sodium phosphate, 0.1 M NaCl, pH 7.5. Molecular weight was determined at several protein concentrations using time-lapse photography (26, 27). SDS disc gel electrophoresis. Polyacrylamide gels (10%) were prepared according to the procedure of Weber and Osborn (281. Samples were run at 5 mA/ gel, and gels were stained for protein with amido black dye. Absorption and circular dichroic spectra. Absorption spectra were obtained with a Cary Model 14 recording spectrophotometer equipped with O-O, 1 and O-l.0 absorbance slide wires using quartz sample cells of l- to lo-mm pathlength. Circular dichroism (CD) was determined with a Cary 61 spectropolarimeter using quartz sample cells of O.l- to lomm pathlength. Units of molar ellipticity, [0], are degrees per squared centimeter per decimole. The sample compartments of both instruments were continuously purged with N, to prevent oxidation of the cobalt enzymes. Sample cells were rendered metal free by soaking for 12 h in a 1:l solution of
508
ARTHUR
concentrated HNOs and H,SO, followed rinsing with metal-free distilled H,O.
by extensive
J. SYTKOWSKI
RESULTS
Enzyme Purification, Assessment of Homogeneity, and Molecular Weight
Isolation and purification of yeast alcohol dehydrogenase from fresh baker’s yeast by the method of Hvidt and Kagi (17) result in an enzyme with a specific activity of 400-425 U/mg after four recrystallizations. Further recrystallizations do not increase specific activity. The crystals obtained at this point are quite stable, and the loss of specific activity is less than 5% in 4 months when suspended in 20 mM sodium phosphate, 60% saturated in (NH4@04, pH 7.1, 4°C. Gel filtration of the enzyme through Sephadex G-200 increases the specific activity to 430-480 U/ mg. Disc gel electrophoresis of this enzyme in the presence of sodium dodecyl sulfate reveals a single band of apparent molecular weight of 37,000 & 3,000, slightly lower than the subunits of human and horse liver alcohol dehydrogenase (29). No other protein species is found. Sedimentation velocity experiments further confirm molecular weight homogeneity, demonstrating a single, narrow, symmetrical boundary. The apparent molecular weight (i&J was determined at several protein concentrations by ultracentrifuge time-lapse photography (see Materials and Methods). A plot of l/M,,, vs protein concentration is linear. Extrapolation to zero protein concentration yields a molecular weight of 149,000. The molar absorptivity (E) at 280 nm, assessed by gravimetric determination of the protein dry weight after dialysis of two 20-mg samples against H,O (see Materials and Methods), was found to be E = 1.83 x lo” M-’ cm2. (A!&% = 1.23 ? 0.01.) Metal Content of YADH
In YADH prepared as described above zinc is the only metal present in stoichiometrically significant amounts as determined by emission spectrography (24). Trace amounts of aluminum and magnesium are variably present while Ba, Ca, Cd, Co, Cr, Cu, Mn, MO, Ni, Pb, and Sn are not detected.
The Zn content of YADH was determined by atomic absorption, atomic fluorescence, and microwave emission spectrometry. Previous comparison of these three methods by analysis of several zinc metalloenzymes of known metal stoichiometry and homogeneity has demonstrated their accuracy, precision, and equivalence (14). Analyses of YADH prepared from fresh baker’s yeast under conditions which eliminate contamination by extraneous metal ions reveal 4.0 +- 0.3 gatom of zinc per mole of enzyme (Table I). No significant differences are found among the three analytical methods. YADH prepared in this laboratory from commercially dried yeast, the conditions and time of storage of which are unknown, has a slightly lower specific activity (400 U/mg) but similarly contains 4.2-4.3 g-atom of Zn/mol. Samples of commercially available enzyme consistently exhibit lower specific activities (260-400 U/mg) and, more important, their zinc contents are higher, ranging from 5.7 to 7.1 g-atom/m01 (Table I), suggesting contamination with adventitious metal atoms (see Discussion). There is no apparent correlation between the specific activities and zinc contents of these commercial enzyme samples. Efforts to obtain either a sample of BoehringerMannheim yeast from which enzyme could be prepared or details of their commercial preparative procedure were unsuccessful. Coenzyme Binding
The binding of NADH to YADH results in a slight hypsochromic shift of the NADH absorption spectrum. The new maximum is centered at 335 nm and exhibits a molar absorptivity, esX5,of 5880 M-’ cm-’ compared to es4,,= 6220 M-’ cm-’ for uncomplexed NADH (Fig. 1). This YADH *NADH complex is optically active with a negative circular dichroic extremum at 330 nm (Fig. 2, upper panel). A circular dichroism titration demonstrates an NADH/YADH binding stoichiometry of 4:l (Fig. 2, lower panel), identical to the zinc stoichiometry. These results are similar to those reported by Temler and Kagi (8). The 330~nm extremum has a maximum molar ellipticity, /&, of - 1.44 x 10” deg *cm2*dmol-’ , compared to that
PROPERTIES
OF YEAST
ALCOHOL TABLE
509
DEHYDROGENASE
I
SPECIFIC ENZYMATIC ACTIVITY AND ZINC CONTENT OF YEAST ALCOHOL DEHYDROGENASE FROM VARIOUS SOURCES Enzyme
Specific activity (LJ/mg)
source
Zinc content”
(g-atom/m01
Atomic absorption
Atomic fluorescenceb
of YADH) Microwave emission
Baker’s yeast preparations’ I(F)-12/74-l I(F)-l/75-2 I(F)-6/75-3 II(F)-8/75-4 III(F)-6/76-5 IV(F)-8/76-6 I(P-L&8/76-6 II(P-L)-8/76-8
460 430 450 480 460 470 400 400
4.0 3.9 4.3 3.7 4.0 4.0 4.2 4.3
4.1 4.1 4.2 3.8 3.9 4.0 4.4 4.2
3.9 4.1 4.1 3.9 3.9 4.0 4.2 4.4
Commercial YADH” B/M 7035436 B/M 7245437 B/M 7315537 B/M 1056138 B/M(X) 7235521 B/M(X) 7285321 S 84C-8570 S 114C-7830 S 45C-8241 W 35M 995
330 280 365 300 390 400 270 280 260 260
5.7 6.2 6.6 6.9 7.1 6.9 6.4 6.2 6.2 6.5
6.0 6.0 6.6 7.0 7.1 7.0 6.3 6.2 6.1 6.7
5.9 6.1 6.6 7.1 7.1 6.8 6.5 6.2 6.4 6.8
n All zinc contents shown are the averages of three to five measurements and replicate measurements all fall within a range of 25%. b Data kindly provided by Dr. Claude Veillon. c As prepared in this laboratory from baker’s yeast (see Materials and Methods). Roman numerals denote yeast batch. (F) = Federal Yeast Corp. (air dried); I(P-L) = P-L Biochemicals “active dried” (Lot No. 3850); II(P-L) = P-L Biochemicals “compressed baker’s” (Lot No. 3217). I(P-L) and II(P-L) were dried by the supplier. d B/M = Boehringer-Mannheim; S = Sigma Chemical Co.; W = Worthington Biochemicals. X denotes crystalline suspension; all other samples are lyophilized powders,
.-. : e w
4--
/’
‘\\ \
\
/I 2-- ’
-/\ 0
\\
//’
300
340 WAVELENGTH.
\\
\\
\\
\\
‘\
380‘ nm
FIG. 1. Effect of YADH binding on absorption spectrum of NADH. Free NADH, 340 pM c---j; NADH, 340 PM, in the presence of 190 FM YADH which equals 760 pM active site concentration (---). Spectrum of NADH-YADH complex determined vs 190 PM YADH blank. Buffer, 20 mM Na-phosphate, pH 7.5, 23°C.
of the NADH complex with horse liver alcohol dehydrogenase, OS85= -8.6 x lo4 deg. cm” *dmolk’, in which only 2 mol of coenzyme is bound (30). The ellipticities per mole of NADH of the YADH *NADH and LADH *NADH complexes are nearly identical, -3.6 x lo4 and -4.2 x 10” deg. cm’ *dmol-‘, respectively, suggesting similar modes of coenzyme binding in the two enzymes, consistent with, deductions made from nuclear magnetic resonance studies of NADH binding to YADH (31). The apparent equilibrium constant, K alw7 for NADH binding was assessed by CD titration of 2.3 PM YADH. A plot of l/ (0) vs l/[NADHh: is linear and reveals K aPP= 26 PM (Fig. 31, in close agreement with previously reported values (2, 8). The stoichiometry of NADH binding
510
ARTHUR 300 I
ri
350 I
J. SYTKOWSKI
I
4.5 for the peak and trough, respectively, in agreement with the values obtained by CD titration (see above). Conditions
OIY
4
12 NADH/YADH.
20
28
mols/mol
FIG. 2. Circular dichroism titration of [(YADH)Zn,l with NADH. Upper panel: The magnitude of the negative circular dichroic trough at 330 nm increases as NADH is added. Aliquots of 12.3 or 60 mM NADH (5-50 ~1) added sequentially. Lower panel: Extrapolation of titration data indicates binding stoichiometry of 4 mol of NADH/mol of YADH. Enzyme, 0.18 mM; 20 mM sodium phosphate, pH 7.5, 4°C. Units of molar ellipticity, 101, are degrees per squared centimeter per decimole.
of Zinc Exchange
The advantages of ‘j5Zn replacement of the stable zinc atom in the active site of a metalloenzyme have been clearly demonstrated (11). The determination of optimal conditions for Zn $ 65Zn exchange in YADH necessarily includes an examination of the effect of pH, which has been shown to be of importance both to metal exchange in enzymes (33) and to acid-assisted dechelation in model systems (34). Dialysis of YADH against 20 mM sodium phosphate in the absence of any added metal ions results in a time- and pH-dependent loss of activity at pH 4.6 and below (Fig. 5A). However, the addition of 047
s $
045 r-
0
20
60
40 EFFLUENT,
ml
FIG. 4. Stoichiometry of NADH binding to [(YADH)Zn,l by gel filtration method of Hummel and Dreyer (32). NADH, 680 PM; enzyme, 30 nmol applied in 50 ~1 of buffer; Na-phosphate, 20 mM, pH 7.5, 23°C; Bio-Gel P-4 (100-200 mesh), 1.5 x 26 cm, 0.8 ml/min, 500-~1 fractions; l-mm pathlength cuvette. FIG. 3. Double reciprocal plot of NADH circular dichroism titration of [(YADH)Zn,]. Titration of relatively low concentration of enzyme, 2.3 pM, with aliquots of 212 PM NADH. The intercept on the abscissa of l/(e) vs l/[NADH&,, is - l/K,,, constant of where Kapp = 26 PM, the dissociation enzyme-coenzyme complex (56).
was also examined by the gel filtration method of Hummel and Dreyer (32) (Fig. 4). Employing molar absorptivities at 340 nm of 5710 M-’ cm-’ for the complexed NADH and 6220 M-’ cm-’ for the free NADH (Fig. 11, calculation of the micromoles of NADH bound per micromole of YADH yields stoichiometries of 3.7 and
FIG. 5. (A) pa-dependent inactivation of YADH in the absence of added Zn. pH 5.0 (a), 4.6 (m), 4.4 (A), and 4.2 (r). (B) Zn protection of pH-dependent inactivation. pH 5.0 (0), 4.6 0, 4.4 (A), and 4.2 (v); 0.4 mM ZnSO1. Enzyme, 0.18 mM; 20 mM sodium at phosphate, 4°C. V,, control enzymatic activity zero time; V, observed activity at specified time.
PROPERTIES
OF YEAST
ALCOHOL
0.4 mM Zn2+ prevents this inactivation (Fig. 5B). These results are strikingly similar to those obtained by Drum (33) in his initial studies of zinc exchange in horse liver alcohol dehydrogenase and, hence, suggest possible conditions for metal exchange in YADH. Below pH 4.1, the enzyme rapidly denatures and is only 40% active after 6 h even in the presence of 0.4 mM Zn2+. The zinc content of YADH after dialysis against Zn2+ under these conditions is identical to that of the starting material, demonstrating that YADH does not acquire adventitious metal atoms when treated in this manner. The specific enzymatic activity of the resultant enzyme is 430-480 U/mg, identical to that exhibited by the homogeneous enzyme freshly prepared from baker’s yeast4 “VJinc Exchange
in YADH
The four zinc atoms of native [(YADH)Zn,] are replaced by ‘j”Zn under conditions of equilibrium dialysis at 4°C. Ten milliliters of [(YADH)Zn,l, 50 mg/ml, in 20 mM sodium phosphate, pH 7.5, is dialyzed against 1000 ml of 1 mM ssZnz+, 0.1 M sodium acetate, pH 4.20. This affords optimal yield of radiolabeled enzyme within a convenient time period. At various intervals aliquots of enzyme are removed from the dialysis bag and are analyzed for fi”Zn content. After equilibrium is reached, the exchange is terminated by changing the buffer to 20 mM sodium phosphate, pH 7.5. The sample is dialyzed for several hours against two additional changes of this buffer and centrifuged (6O,OOOg,30 min, 4°C) to remove precipitated material. The 4 g-atom of intrinsic zinc of the enzyme have exchanged fully after 8-9 h of dialysis against ““Zn (Fig. 6). During the exchange approximately 4 Enzyme obtained after only two (NH&SO, recrystallizations (see Materials and Methods) has a specific activity of 310-330 U/mg. Dialysis of such partially purified enzyme against Znz+ in this pH range also yields homogeneous enzyme of 430-480 U/mg containing 4 g-atom of Zn per mole after precipitated impurities are removed by centrifugation. Hence, this dialysis step can be used as a method for final purification eliminating two recrystallizations and gel filtration.
511
DEHYDROGENASE
30% of the enzyme precipitates, resulting in a final yield of 70%. As with LADH (33, 351, the rate of metal exchange in YADH is inversely related to protein concentration. Hence, this precipitation of enzyme during exchange results in progressively faster exchange. Thus, the apparent exchange data do not conform to simple firstorder kinetics. All four zinc atoms of YADH exchange identically in acetate or phosphate buffers, indicating that the four zinc atoms are equivalent. This is in marked contrast to horse liver alcohol dehydrogenase where the presence of these buffers differentiates between two noncatalytic and two catalytic zinc atoms (10, 11, 35). The zinc content, specific enzymatic activity, and coenzyme binding of “SZn-labeled yeast alcohol dehydrogenase, [(YADH)““Zn,l, are identical to those of the native enzyme (Table II), demonstrating that “;‘Zn has
FIG. 6. ““Zn exchange in YADH. ““Zn content by y emission spectrometry (0); 1 mM ‘“Zn’+, 20 mM sodium acetate, pH 4.2, 4°C. TABLE II ZINC CONTENT, SPECIFIC ENZYMATIC ACTIVITY, AND COENZYME BINDING OF I(YADH)““Zn,l [(YADHI““Zn,] preparations
BRL BRL BRL BRL
75-1 75-2 76-1 76-2
Total zinc content” (g-atom/ mol) Atomic absorption
y emission
4.1 4.0 4.2 3.9
4.2 3.9 4.2 4.0
Specitic activity W/ wd
NADHI YADHb (mol/ mol)
460 450 480 470
4.0 3.9 ND ND
(’ Total zinc content determined by both atomic absorption spectrometry and y emission spectrometry of “sZn. Results are 25%. * Circular dichroism titration; ND = not determined.
512
ARTHUR
indeed replaced the catalytically native zinc atoms. Cobalt Exchange
J. SYTKOWSKI
essential
in YADH
Substitution of cobalt for zinc in YADH is accomplished by dialysis of [(YADH) 65Zn41, 21 mg/ml, against a loo-fold volume excess of 0.1 M CoCI,, 20 mM sodium acetate, pH 4.50, 4”C, under 1 atm of oxygenfree N,. Two additional changes of the dialysate are employed to prevent back exchange of zinc. At specific intervals aliquots are removed from the dialysis sac, dialyzed free of extraneous metal atoms against 20 mM Tris-HCl, pH 7.5, 4”C, centrifuged to remove precipitated material, and analyzed for zinc, 6sZn, and cobalt. In the course of dialysis the zinc content of the enzyme decreases and cobalt content increases proportionally. Independent determinations of zinc content by atomic absorption spectrometry and y emission spectrometry are virtually identical, and at all times during exchange the total metal content of each sample, C (Zn + Co), is 4.0 + 0.2 g-atomlmol. Hence, the reliability of ““Zn loss as a precise measure of the rate, extent, and site specificity of cobalt replacement is demonstrated. When equilibrium is reached the exchange is stopped and free metal atoms are removed by dialysis of the enzyme against several changes of 20 mM Tris-HCl, pH 7.5, 4°C. The resultant enzyme, [(YADH)Co,], is green and contains 3.9 g-atom of cobalt and only 0.1-0.2 g-atom of zinc/mol. The specific enzymatic activity of the fully cobalt-substituted enzyme is 80 U/mg. This activity, while only 17% of that of the native enzyme, is much higher than can be accounted for by residual zinc (3-5% of that of the native enzyme). In addition, resubstitution of Zn into [ (YADH)Co,] results in a corresponding increase in activity, demonstrating that the reduced activity of [(YADH)CoJ is specifically due to replacement of the catalytically essential Zn atoms with Co and not to enzyme denaturation. Absorption
and Circular
Dichroic
Spectra
[ (YADH)CoJ exhibits absorption maxima at 340 and 627 nm with shoulders at
675-685 and 725-745 nm (Fig. 7). The molar absorptivities (6) are A,,,, (14,800), A627 u>7w, ~6,S+w (1,320), and h725-,45(680). The position and intensity of these maxima are strikingly similar to those exhibited by cobalt-substituted horse liver alcohol dehydrogenase, 1(LADH)Co$o,] (11). Associated with the 340-nm cobalt absorption maximum are two negative circular dichroic extrema centered at 332 and 388 nm and a shoulder at 310 nm with molar ellipticities, [el, of AsI0 G-11,900), A (-21,600), and A38X (-13,000) d$ *cm* *dmol-‘. In addition, a very weak ([Ol - 300-800) negative ellipticity is present at 550-700 nm (not shown) corresponding to the absorption maxima in this region (Fig. 8). DISCUSSION
Over 20 years ago YADH was shown to contain 4 g-atom of catalytically essential zinc per mole (1) and to bind 4 mol of coenzyme (2). Subsequently, Wallenfels and Sund (3, 4) reported stoichiometries of 5:l for metal content and coenzyme binding. Later, several reports confirmed the metal content of 4 g-atom/m01 (6, 7, 15, 36). However, recently both the coenzyme 2 3 PQ * Y
m x 0 1 L
2
0lud
- 1 350
-
0
450
600
700
WAVELENGTH.nm
FIG. 7. Absorption spectrum Tris-HCl, 20 mM, pH 7.5, 4°C.
L-&
-d
of
[(YADH)Co41.
+ 400
W&VELENGTH,
nm
dichroic spectrum of FIG. 8. Circular [(YADH)Co41. Tris-HCl, 20 mM, pH 7.5, 4°C. Units of molar ellipticity, [Ol, are degrees per squared centimeter per decimole.
PROPERTIES
OF YEAST
ALCOHOL
binding and zinc stoichiometries have again come into question. The NADH/ YADH ratio of 3:l reported by Dickinson (37) and by Sanderson and Weiner (38) has been revised downward to 2:l (7), similar to the ratio of 1.751 reported by Leskovac and Pavkov-Pericin (39). In addition, Dickinson’s earlier zinc stoichiometry of 4.3-5.0 g-atom/m01 (7) has also been revised to 7.6 g-atom/m01 for a “relatively impure” enzyme preparation (40), a value similar to the 7.1-7.4 g-atom/m01 reported by Klinman and Welsh (16). These findings of a higher zinc content were subsequent to enunciation of the hypothesis of Branden et al. (13), based on the partial sequence homologies between YADH and LADH, that, as in LADH, two classes of zinc atoms might exist in YADH. Hence, YADH might contain not 4 but 8 g-atom of Zn. This prompted a reinvestigation of the Zn content of homogeneous YADH by means of three highly sensitive and accurate analytical methods, which have confirmed that the intrinsic zinc content is 4 g-atom/m01 (14). YADH readily binds adventitious metal atoms, as was well documented by Wallenfels and Sund (3, 4), Redetzki and Nowinski (41), and Hoch and Vallee (42). The metal stoichiometries of YADH preparations obtained from several commercial sources are variable and in excess of 4 gatom of Zn/mol (Table I). This excess almost certainly reflects adventitiously bound zinc acquired in the course of enzyme isolation. The lack of detailed information regarding the preparative procedures used in these instances precludes a definitive statement regarding the source of contamination (vide infra). Wallenfels and Sund (3, 4) and Sund (5) reduced the excess zinc content of YADH from a total of 35 to 4.7 g-atom per mole by dialysis against EDTA. They suggested that the chemical environment of the enzyme during isolation and even the properties of the yeast culture itself might result in binding of adventitious zinc atoms. Hoch and Vallee (42) showed that the zinc content of a preparation with 5.2 g-atom of zinc per mole was reduced to 4.0 g-atom by dialysis at pH 6.0 while both specific enzymatic activity and the number of
DEHYDROGENASE
513
titrable sulfhydryl groups actually increased, consistent with the demonstrated inhibition of YADH activity by adventitious metal atoms and reactivation upon their removal by chelating agents (5, 41). Recent studies confirm that EDTA removes excess zinc from YADH (14, 16) although under certain conditions of dialysis the enzyme is apparently unstable and loses activity (16). The problem of adventitious metal binding is not unique to YADH, of course, and is an important consideration in the study of all metalloenzymes (43). It is critical that investigations of such enzymes be carried out on well-characterized homogeneous material and that particular attention be given to the prevention of metal contamination. Metal contamination can arise from many sources. Buffer contamination may result from impure reagents or water (including acids or bases used to adjust pH), from pH electrodes, from containers used to prepare and store the buffers, and even from airborne sources. Consequently, solutions must be prepared from materials known to be free of metals or these extraneous metals must be removed either by solvent extraction with a chelating agent or by passage through an ion exchange resin. Even then, it cannot be assumed that the solutions so treated are necessarily rendered free of contaminating metals, since, for example, treatment with ion exchange resins may remove one contaminant and contribute another. Thus, the absence of contamination must be verified by suitable metal analysis. Gel filtration and ion exchange media, and associated equipment used for enzyme purification, must be similarly free from metals. In the present isolation and purification of YADH strict attention was paid to the elimination of all sources of metal contamination. The resultant enzyme is homogeneous, of high specific activity, and contains 4 g-atom of catalytically essential, intrinsically bound Zn per mole of enzyme. This stoichiometry is identical to that found for coenzyme binding and indicates one Zn atom and one coenzyme binding site per subunit. The detailed X-ray crystallographic
514
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analysis of horse liver alcohol dehydrogenase (13) has confirmed the conclusions of previous studies on the nature of the zinc atoms of that enzyme (9, 35) and has further delineated knowledge regarding coordination of its metal atoms. The partial homologies in the amino acid sequences of horse LADH and YADH described previously (12) led to the suggestion that the tetrameric yeast enzyme might contain two firmly bound Zn atoms per subunit, i.e., eight per tetramer, analogous to the catalytic and noncatalytic Zn atoms of the dimeric horse enzyme, or, alternatively, that the cysteinyl residues in the yeast enzyme which are homologous to ligands of the noncatalytic Zn atoms in the horse enzyme may form S-S bonds (13). The results of the present study show that there are only four firmly bound catalytically essential zinc atoms per tetramer in YADH. There is no evidence for a second, firmly bound structural zinc atom per subunit in this enzyme. Apparently the tertiary structure of the region in YADH containing these homologous cysteinyl residues does not permit the firm binding of an additional zinc atom. Importantly, comparison of the now completed amino acid sequence of YADH (44) to that of LADH reveals some striking differences between the two enzymes, especially in that region (residues 90-149) which binds the noncatalytic Zn atom in LADH. In this region only “random similarities” exist between LADH and YADH strongly suggesting that potential Zn ligands in YADH “need not be conserved in space although strictly homologous in primary structure” (45). The replacement of the zinc atoms of native enzymes by other metals with more favorable spectral properties has greatly facilitated the elucidation of the role of metal atoms in the mechanism of action of metalloenzymes (43). In this regard, the chromophoric and paramagnetic cobalt atom has been of particular value in the investigation of carboxypeptidase (461, carbonic anhydrase (47), alkaline phosphatase (48, 491, thermolysin GO), horse LADH (10, 11, 51), leucine aminopeptidase (521, and other enzymes (43). In contrast
to most other metalloenzymes, it has thus far not been possible to prepare stable apo-LADH by exposure to chelating agents, thus rendering it impossible to study the restoration of activity by direct addition of zinc or other metal atoms. Therefore, methods to exchange metals by equilibrium dialysis were devised to replace the native zinc atoms in LADH (33, 35, 53). Similarly, it has not been possible to remove the catalytically essential zinc atoms from YADH without apparently irreversible denaturation of the enzyme. In the present study, the methodological principles developed for metal exchange in horse LADH (33) have been applied to studies of the yeast enzyme, and the conditions for replacement of the native zinc atoms of this enzyme with ““Zn and subsequently with Co have been derived. It is essential that metal exchange be performed with enzyme of high purity and specific activity and of known, uniform metal stoichiometry, criteria which demanded rigorous investigation of the methods for the isolation, purification, and characterization of YADH prior to undertaking exchange studies. The enzyme prepared and purified from fresh yeast as described here meets these criteria. Under all conditions investigated the exchange properties of the four Zn atoms in YADH were found to be the same. All four atoms exchange identically in either the phosphate or acetate buffers used, in striking contrast to LADH, where the differences in exchange characteristics distinguish between the catalytic and noncatalytic Zn atoms (11, 35). This suggests that the four Zn atoms in YADH are equivalent, and it may then be presumed that they are located in identical environments within the enzyme, i.e., one zinc atom at each active site. Importantly, the exchange is pH dependent over a very critical, narrow range (Fig. 5), approximately 1 pH unit lower than that of the horse enzyme, and is extremely sensitive to slight deviations in hydrogen ion concentration. [ (YADHjG5Zn,l has the same specific activity metal content and coenzyme binding properties as the native en-
PROPERTIES
OF YEAST
ALCOHOL
zyme. Using this radiolabeled enzyme, it is now possible to substitute other metal atoms, such as cobalt, for zinc in YADH and to be certain of the site specificity and extent of such substitution, as shown for LADH (11). Since YADH readily binds adventitious metal atoms (vide supra), the location of any “replacement” atoms in the enzyme is open to serious question unless the simultaneous loss of the intrinsic, catalytically essential zinc atoms is unambiguously demonstrated by such quantitative, site-specific means. Previously, YADH containing metal atoms other than Zn has been obtained by isolation of the enzyme from yeast grown in medium rich in the respective metal. Curdel and Iwatsubo (15) prepared a green enzyme with approximately 1.2 g-atom of Co and 2.3 g-atom of Zn/mol. The absorption spectrum of this enzyme exhibited broad maxima at 620, 670, and 710 nm qualitatively similar to those obtained in the present study. Unfortunately, a specific activity for this enzyme was not reported. Another example of metal replacement in YADH by biosynthetic means is the isolation of a manganese YADH by Coleman and Weiner (6). This enzyme contained 3.7 * 0.6 g-atom of Mn/mol and no detectable Zn. The enzyme was far less stable than the corresponding Zn enzyme and, hence, it was necessary to store it in the presence of 20% sucrose and 0.1% mercaptoethanol. Also, 1 mM EDTA inactivated the Mn enzyme but not the Zn enzyme. Recently, a series of “hybrid enzymes” containing 1.0-2.9 g-atom of Co and 3.8-5.6 g-atom of Zn per mole of YADH have been prepared by dialysis of enzyme containing 7.0 g-atom of Zn against Co”+ (16). The specific locations of these replacement metal atoms in the enzyme have not yet been ascertained. The present study is the first report of the preparation of cobalt-substituted yeast alcohol dehydrogenase in which the site specificity and extent of the cobalt substitution are precisely delineated. The resultant fully substituted enzyme, [(YADH)Co,], contains 3.9 g-atom of Co and only 0.1-0.2 g-atom of Zn per mole. The data indicate that in this enzyme Co
DEHYDROGENASE
515
has specifically replaced the four intrinsic, catalytically essential Zn atoms. By analogy with the two catalytic, active site metal atoms of horse liver alcohol dehydrogenase, a dimer, the four intrinsic metal atoms of YADH, a tetramer, might be presumed also to lie in the active sites of the enzyme, one per subunit. Hence, the Co atoms of [(YADH)Co,l would serve to reflect events occurring at this locus. This is supported by the change in specific enzymatic activity which ensues from this metal replacement, similar to that observed upon Co replacement of the catalytic but not the noncatalytic Zn atoms of horse LADH (11). Moreover, the absorption spectrum of [(YADH)Co,l strongly suggests similarities in coordination of Co in this enzyme with the cobalt atoms of substituted enzyme, the horse l(LADH)Co2Co,l. The 340-nm absorption maximum of [(YADH)Co,l exhibits a molar absorptiviity (e) of 15,800, i.e., 395O/Co atom, virtually identical to 3750/catalytic Co atom observed in [ (LADH)Co,Co,]. The position and intensity of this maximum likely represent charge transfer between Co and sulfhydryl ligands (54, 55) which have been demonstrated to coordinate the active site metal in LADH (13) and which amino acid sequence homology suggests are present in YADH (44, 45). The visible absorption maxima of l(YADH)Co,] and l(LADH)Co,Co,l are also similar in position and intensity and are consistent with distorted tetrahedral coordination geometries in both enzymes (55). In contrast, the circular dichroic spectrum of [(YADH)Co,l is strikingly different from that of [(LADH)Co,Co,l (A. J. Sytkowski and B. L. Vallee, in preparation). Although a detailed interpretation of these CD spectral characteristics is not possible at present, they clearly are indicative of the asymmetry inherent in the coordination complex of the catalytically essential cobalt atom. These data establish the physicochemical, analytical, and enzymatic properties of highly purified YADH as well as the conditions for ““Zn and cobalt exchange. This knowledge now permits the detailed investigation of the role of the catalyti-
516
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tally essential metal atoms in the enzyme. Taken together, these methods illustrate a general approach to the elucidation of the functional and structural roles of metal atoms in metalloenzymes (57). ACKNOWLEDGMENTS The author wishes to thank Dr. J. L. Bethune and Dr. W. Dafeldecker for valuable assistance and discussions of the analytical ultracentrifuge data, Dr. Gerald M. Alter for advice on the circular dichroism titrations, and Dr. Claude Veillon for his participation in the early phase of this work (14). REFERENCES 1. VALLEE, B. L., AND HCICH, F. L. (1955) PFOC. Nat. Acad. Sci. USA 41, 327-338. 2. HAYES, J. E., JR., AND VELICK, S. F. (1954) J. Biol. Chem. 207, 225-244. 3. WALLENFELS, K., AND SUND, H. (1957) Biochem. z. 329, 31-40. 4. WALLENFELS, K., AND SUND, H. (1957) Biochem. z. 329, 59-74. 5. SUND, H. (1957) Ph.D. thesis, Hamburg. 6. COLEMAN, P. L., AND WEINER, H. (1973) Biochemistry 12, 3466-3472. 7. DICKINSON, F. M. (1974) EUF. J. Biochem. 41, 31-36. 8. TEMLER, R. S., AND K;~GI, J. H. R. (1973) Abstracts, IXth International Congress on Biochemistry, Abstract 2r4, p. 109. 9. DRUM, D. E., AND VALLEE, B. L. (1970) Biochemistry 9, 4078-4086. 10. SYTKOWSKI, A. J., AND VALLEE, B. L. (1975) Biochem. Biophys. Res. Commun. 67, 14881493. 11. SYTKOWSKI, A. J., AND VALLEE, B. L. (1976) PFOC. Nat. Acad. Sci. USA 73, 344-348. 12. JBRNVALL, H. (1973) Proc. Nat. Acad. Sci. USA 70, 2295-2298. 13. BRKND~N, C.-I., JGRNVALL, H., EKLUND, H., AND FURUGREN, B. (1975) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 11, Part A, pp. 103-190, Academic Press, New York. 14. VEILLON, C., AND SYTKOWSKI, A. J. (1975) Biothem. Biophys. Res. Commun. 67, 1494-1500. 15. CURDEL, A., AND IWATSUBO, M. (1968) FEBS Lett. 1, 133-136. 16. KLINMAN, J. P., AND WELSH, K. (1976)Biochem. Biophys. Res. Commun. 70, 878-884. 17. HVIDT, A., AND K;~GI, J. H. R. (1963) Compt. Rend. Trav. Lab. Carlsberg 33, 497-534. 18. THIEFLS, R. (1957) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 5, pp. 273-335, Wiley-Interscience, New York. 19. ALVAREZ, R. A., PAULSEN, P. J., AND KELLEHER, D. E. (1969) Anal. Chem. 41, 955-958. 20. FUWA, K., PULIDO, P., MCKAY, R., AND VALLEE,
B. L. (1964) Anal. Chem. 36, 2407-2411. 21. CLYBURN, S. A., SERIO, G. F., BARTSHMID, B. R., EVANS, J. E., AND VEILL~N, C. (1975) Anal. Biochem. 63, 231-240. 22. VEILLON, C. (1976) in Trace Analysis, Vol. 46, (Winefordner, J. D., ed.), pp. 123-181, WiieyInterscience, New York. 23. KAWAGUCHI, H., AND VALLEE, B. L. (1975)Anal. Chem. 47, 1029-1034. 24. VALLEE, B. L. (1955) Advan. Protein Chem. 10, 317-384. 25. HOCH, F. L., AND VALLEE, B. L. (1953) Anal. Chem. 25, 317-320. 26. BETHUNE, J. L. (1970) Biochemistry 9, 27372744. 27. SIMPSON, R. T., AND BETHUNE, J. L. (1970) Biochemistry 9, 2745-2750. 28. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 29. LANGE, L. G., SYTKOWSKI, A. J., AND VALLEE, B. L. (1976) Biochemistry 15, 4687-4693. 30. SYTKOWSKI, A. J., AND VALLEE, B. L. (1977) Biochemistry, in press. 31. SLOAN, D. L., AND MILDVAN, A. S. (1974) Biochemistry 13, 1711-1718. 32. HUMMEL, J. P., AND DREYER, W. J. (1962) Biochim. Biophys. Acta 63, 530-532. 33. DRUM, D. E. (1967) Ph.D. thesis, Massachusetts Institute of Technology. 34. WILKINS, R. G. (1974) The Study of Kinetics and Mechanism of Reactions of Transition Metal Complexes, Allyn and Bacon, Boston. 35. DRUM, D. E., LI, T.-K., AND VALLEE, B. L. (1969) Biochemistry 8, 3792-3797. 36. LESKOVAC, V., TRIVIC, S., AND LATKOVSKA, M. (1976) Biochem. J. 155, 155-161. 37. DICKINSON, F. M. (1970) Biochem. J. 120, 821830. 38. SANDERSON, J. A., AND WEINER, H. (1973) Fed. Proc. 32, 543. 39. LESKOVAC, V., AND PAVKOV-PERICIN, D. (1975) Biochem. J. 145, 581-590. 40. DICKENSON, C. J., AND DICKINSON, F. M. (1976) Biochem. J. 153, 309-319. 41. REDETZKI, H. E., AND NOWINSKI, W. W. (1957) Nature (London) 179, 1018-1019. 42. HOCH, F. L., AND VALLEE, B. L. (1959) in A Symposium on Sulfur in Proteins (Benesch, R., ed.), pp. 245-265, Academic Press, New York. 43. VALLEE, B. L., AND WACKER, W. E. C. (1970) in The Proteins (Neurath, H., ed.), 2nd ed., Vol. 5, Academic Press, New York. 44. JGRNVALL, H. (1977) Eur. J. B&hem. 72, 425442. 45. JGRNVALL, H. (1977) EUF. J. Biochem. 72, 443452. 46. COLEMAN, J. E., AND VALLEE, B. L. (1960) J. Biol. Chem. 235, 390-395.
PROPERTIES
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ALCOHOL
47. LINDSKOG, S. (1970) in Structure and Bonding, Vol. 8, (Hemmerich, P., Jorgensen, C. K., Neilands, J. B., Nyholm, R. S., Reinen, D., and Williams, R. J. P., eds.), pp. 153-196, Springer-Verlag, New York. 48. SIMPSON, R. T., AND VALLEE, B. L. (1968) Biochemistry 7, 4343-4350. 49. ANDERSON, R. A., KENNEDY, F. S., AND VALLEE, B. L. (1976) Biochemistry 15, 3710-3716. 50. HOLMQUIST, B., AND VALLEE, B. L. (1974) J. Biol. Chem. 249, 4601-4607. 51. DRUM, D. E., AND VALLEE, B. L. (1970) Biochem. Biophys. Res. Commun. 41, 33-39. 52. THOMPSON, G. A., AND CARPENTER, F. H. (1976) J. Biol. Chem. 251, 1618-1624. BE53. DRUM, D. E., HARRISON, J. H., LI, T.-K.,
54.
55. 56.
57.
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THUNE, J. L., AND VALLEE, B. L. (1967) Proc. Nat. Acad. Sci. USA 57, 1434-1440. FOSTER, M. A., HILL, H. A. O., AND WILLIAMS, R. J. P. (1970) in Chemical Reactivity and Biological Role of Functional Groups in Enzymes, Biochemical Society Symposium No. 31 (Smellie, R. M. S., ed.), pp. 187-202, Academic Press, London. GARBETT, K., PARTRIDGE, G. W., AND WILLIAMS, R. J. P. (1972) Bioinorg. Chem. 1, 309-329. JOHANSEN, J. T., KLYOSOV, A. A., AND VALLEE, B. L. (1976) Biochemistry 15, 296-303. SYTKOWSKI, A. J., AND VALLEE, B. L. (1977) in Pharmacology of Ethanol (Majchrowicz, E. and Noble, E. P., eds.), Plenum Press, New York, in press.
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BIOCHEMISTRY
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184, 505-517 (1977)
Metal Stoichiometry, Coenzyme Binding, and Zinc and Cobalt Exchange in Highly Purified Yeast Alcohol Dehydrogenasel ARTHUR J. SYTKOWSKI* Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts 02115 Received
May 27, 1977; revised
August
12, 1977
Yeast alcohol dehydrogenase, purified from baker’s yeast under conditions which exclude contamination by extraneous metal ions, is homogeneous by analytical ultracentrifugation and disc gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a molecular weight of 149,000 as determined by ultracentrifugation time-lapse photography and exhibits specific activities of 430 to 480 U/mg. Zinc analysis by three independent, highly sensitive methods, i.e., atomic absorption spectrometry, atomic fluorescence spectrometry, and microwave-induced plasma emission spectrometry, demonstrates 4 g-atom of catalytically essential Zn per mole of enzyme. No other metal atoms are present in stoichiometrically significant quantities as assessed by emission spectrography. The stoichiometry of coenzyme binding, 4 mol of NADH/mol of enzyme, is identical to that of zinc, consistent with one coenzyme binding site and one zinc atom per enzyme subunit. Conditions for exchange of the four catalytically essential zinc atoms with ““Zn have been developed. These atoms exchange identically under all conditions examined. The resultant radiolabeled enzyme, [(YADH)““Zn,], has the same metal content, specific enzymatic activity, and coenzyme binding properties as the native enzyme. The ““Zn of this enzyme serves to monitor the extent and site specificity of cobalt replacement. The fully cobalt-substituted enzyme, [(YADH)Co,], has a specific activity of 80 U/mg, 17% that of the Zn enzyme, and exhibits absorption and circular dichroic spectra which are consistent with coordination by one or more sulfur ligands in a distorted tetrahedral geometry.
Yeast alcohol dehydrogenase was first demonstrated to be a zinc metalloenzyme by Vallee and Hoch (11, who found a stoichiometry of 4.1 g-atom of catalytically essential zinc per mole of enzyme, employing both emission spectrographic and microchemical spectrophotometric methods for metal analysis. This stoichiometry is identical to that reported for NADH binding to the enzyme (2) and is consistent with one active site zinc atom and one coenzyme binding site per subunit.
Subsequently, Wallenfels and Sund (3, 4) and Sund (5) documented in great detail the ready acquisition of adventitious zinc by YADH.3 They showed that the enzyme could bind up to 35 g-atom of Zn per mole and that the excess Zn could be removed by various methods including recrystallization and dialysis against EDTA. Recently, Coleman and Weiner (6) isolated YADH from yeast grown in the presence of zinc or manganese and found 3.7 -r- 0.6 g-atom of either metal per mole of enzyme, a stoichiometry consistent with earlier re-
’ This work was supported by Grant-in-Aid GM15003 from the National Institutes of Health of the Department of Health, Education, and Welfare. 2 Special Fellow of the National Institute of Genera1 Medical Sciences, National Institutes of Health. Present address: Department of Pediatrics, Harvard Medical School, Children’s Hospital Medical Center, Division of Hematology-Oncology, 300 Longwood Avenue, Boston, Massachusetts 02115.
pOrtS.
3 Abbreviations used: YADH or [(YADH)Zn,], native yeast alcohol dehydrogenase; [ (YADH)““Zn,], radiolabeled yeast alcohol dehydrogenase; l(YADH)Co,], cobalt-substituted yeast alcohol dehydrogenase; NADH, nicotinamide adenine dinucleotide, reduced form; CD, circular dichroism; LADH, native horse liver alcohol dehydrogenase. 505
Copyright All rights
0 1977 by Academic Press, Inc. of remoduction in anv form reserved.
TSSN
nnnR-RRfii
506
ARTHUR
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Preliminary studies in this laboratory with YADH from commercial sources revealed zinc stoichiometries in excess of 4 g-atom per mole and variable specific activities. A reassessment of coenzyme binding by Dickinson (7) resulted in an NADH/ YADH stoichiometry of 2:1, in contrast to 4:l (2, 8) or 51 (4) reported earlier. The elucidation of two classes of zinc atoms, catalytic and noncatalytic, in horse liver alcohol dehydrogenase (9-11) and partial sequence homologies between YADH and LADH (12) suggested to Branden et al. (13) that the yeast enzyme might contain 8 g-atom of Zn per mole. These considerations prompted a careful reinvestigation of the intrinsic zinc content of well-characterized YADH by three highly sensitive analytical methods (14). Despite the accumulated evidence that homogeneous enzyme contains only 4 g-atom of intrinsic Zn per mole (1, 3, 6, 14, 15), reports of higher metal contents continue to appear (16). The present study describes the physicochemical properties, specific enzymatic activity, metal content, and coenzyme binding characteristics of YADH prepared under rigorously controlled conditions. The results confirm that there are 4 gatom of catalytically essential Zn per mole of YADH and that the enzyme binds 4 mol of coenzyme. In addition, the native Zn atoms have been replaced first by 65Zn and subsequently by Co resulting in [(YADH)‘j5Zn,l and [ (YADH)CoJ, respectively. The cobalt-substituted enzyme is catalytically active and contains a sensitive spectral probe which permits the investigation of the intrinsic metal atoms by a variety of spectroscopic techniques. MATERIALS
AND
METHODS
Yeast. Fresh baker’s yeast was obtained from Federal Yeast Corp. as l-lb cakes, which were finely crumbled and spread in a uniform l-cm layer onto fiberglass window screens covered with two layers of grade 50 cotton cheesecloth. The screens were stacked horizontally with a X-cm space between them, and the yeast was allowed to air dry for 7 days at 22°C in an air-conditioned room with continuous air circulation. Dried yeast not used immediately was sealed in polyethylene bags and stored at
4°C for not longer than a few weeks (17). For comparative purposes, dried yeast was also obtained from P-L Biochemicals in two forms: “baker’s active-dried” and “compressed baker’s” YADH. Commercial preparations of enzyme were obtained either as a lyophilized powder or as a crystalline suspension from Boehringer-Mannheim Corp. and as lyophilized powders from Sigma Chemical Co. and Worthington Biochemical Corp. Reagents and purification. Phosphate buffers were prepared from analytical reagent grade Na2HP0, (Fisher). A 0.5 M stock solution was adjusted to pH 7.5 with HCl and adventitious metals were removed by solvent extraction with 0.01% dithizone in CCL (18). Other buffer concentrations and pH values were attained by appropriate dilution of this stock with metal-free water followed by pH adjustment. Sodium acetate buffer solutions were prepared similarly from a 1 M stock solution of ultrapure NaC2H,01 (anhydrous form) (Alfa Products, Ventron Corp.). Metal-free water was obtained by distillation followed by passage through two mixed-bed ion exchange resin cartridges (Cole-Palmer) in series and stored in a sealed polyethylene container. Air venting into the storage container on withdrawal was passed through a O.&pm filter to prevent airborne contamination. Water prepared and stored in this manner was found to be free of metals when analyzed by emission spectrography, microwave emission spectrometry, and atomic fluorescence spectrometry, down to levels of at least 10e4 pg/ml. Metal-free hydrochloric acid used for pH adjustment of buffers was prepared from metalfree water in cleaned (see below) polypropylene containers by isothermal distillation (19). Metal ion solutions for the exchange experiments were made from 1 M stock solutions of spectrographically pure ZnSO,. 7H,O or CoC12.6H10 (Johnson-Matthey Chemicals, Ltd.), which were analyzed for metal content. A saturated solution (25°C) of (NH&SO, (ultrapure, special enzyme grade, Schwarz/Mann) was prepared in metal-free water, and the pH was adjusted to 7.0 with isothermally distilled NH,OH. Sodium pyrophosphate buffer (0.1 M, pH 8.8) and 1 mM EDTA solutions (tetrasodium salt, pH 8.0) were made from analytical reagent grade materials. pNADH (grade III) and fi-NAD+ (grade III) were obtained from Sigma Chemical Co. Glassware. Whenever possible, plastic containers were employed and the use of glassware was avoided. Glassware was cleaned by soaking for several hours in a 1:l solution of HN03/H2S04, rinsing with metal-free water, soaking for 24 h in a 3% solution of Count-Off decontaminant/detergent (New England Nuclear), and then thoroughly rinsing with metal-free water. Plastic ware (polyethylene, polypropylene, polystyrene) was obtained new, rinsed in tap water to remove dust, soaked in 3%
PROPERTIES
OF YEAST
ALCOHOL
Count-Off for 24 h, and then thoroughly rinsed with metal-free water. After use, the plastic vessels were cleaned by repetition of the same procedure without exposure to highly reactive acids which damage the hydrophobicity of the surface. This procedure rendered these plastic materials completely free of any detectable metal comtamination. Dialysis tubing. Cellulose dialysis tubing (Vs-in. flat width) was prepared by heating cut lengths to 70°C in four changes of metal-free water. They were then transferred to a plastic container, rinsed in two changes of metal-free water, and stored at 4°C in metal-free water. All dialysis tubing, plastic closure clips, magnetic stirbars, etc., were handled with disposable polyethylene gloves (Cole-Parmer, Chicago) to prevent contamination. Enzyme purification. YADH was prepared from dried yeast according to the method of Hvidt and Kagi (17). Using fresh yeast, enzyme crystals having a silken sheen readily appeared at 25% saturation with ammonium sulfate. The specific enzymatic activity was 310-330 U/mg after two recrystallizations and 400-425 U/mg after four recrystallizations (see Results). Further purification was achieved by gel filtration through Sephadex G-200 (Pharmacia) in a 2.5 x 50-cm column, 0.24 ml/min ascending, 20 mM sodium phosphate, pH 7.5. Fractions (1.4 ml) having the highest uniform specific enzymatic activity were combined and concentrated by pressure dialysis (Diaflo PM-10 membrane, Amicon). Membranes were soaked for 24 h in several changes of metal-free water prior to use. Metal analysis. Three sensitive analytical systems for metal determinations were used in this investigation: atomic absorption spectrometry with total consumption, flame atomization and a longpath absorption tube (20), atomic fluorescence spectrometry with continuous sample introduction, and graphite furnace atomization (21, 221, and low-pressure, microwave-induced plasma emission spectrometry employing microvolume sampling (23). In addition, an emission spectrograph (Jarrell-Ash) served to determine the simultaneous multielement composition in ascertaining the presence and stoichiometries of metals other than zinc in YADH (24). All metal determinations reported are the averages of three to five measurements, and replicate measurements all fall within a range of 25%. Radioactivity measurements. The 6SZn radioisotope was measured by y emission spectrometry (Model 1185, Searle Analytic, Inc.). Sufficient counts were accumulated for each measurement to assure r2% reproducibility in the counting statistics at the 95% confidence level. BSZn (sp act = l-10 Ci/g) (New England Nuclear) was added to the buffers used for exchange experiments at a level of 0.1 mCi/lOO ml. Enzymatic actiuity. Activity was determined spec-
DEHYDROGENASE
507
trophotometrically by measuring the rate of formation of NADH at 340 nm and 25°C (1). The specific enzymatic activity reported herein is expressed in units (U) per milligram of protein, where 1 U = 1 +mol of NADH formed per minute. The concentrations of NADH and YADH were determined spectrophotometrically using extinction coefficients of A$: = 6.22 and&:‘,‘,” = 1.23, respectively (see Results). The 3.0-ml reaction mixture contained 5 Fmol of NAD+ and 1 mmol of ethanol in 16.7 mM pyrophosphate buffer, pH 8.8. The assay was initiated with 0.5-l pg of enzyme. Dry weight determinations. The dry weight (25) of protein solutions of known volume and the accurately measured absorbance at 280 nm were established for homogeneous YADH as follows. Samples were dialyzed at 4°C vs several changes of metalfree water, the pH of which had been adjusted to -8 with NH,OH. The absorbance at 280 nm was measured carefully and known volumes were transferred by calibrated pipet into constant-weight beakers, as were similar volumes of the final dialysis buffer for a blank determination. The samples were frozen, lyophilized for 24 h, and weighed on a semimicroanalytical balance (calibrated with N.B.S. Class M weights). They were then heated at 110°C for 1 h, cooled for 2 h, and weighed. This latter process was repeated over several cycles to verify that all the water had been removed by lyophilization. All containers were handled in accordance with standard gravimetric procedures to prevent any surface contamination which might alter their weights. Analytical ultracentrifugation. Sedimentation velocity experiments were carried out at 56,000 rpm at 21°C in a Spinco Model E ultracentrifuge (Beckman), at protein concentrations of 5-10 mg/ml in 20 mM sodium phosphate, 0.1 M NaCl, pH 7.5. Molecular weight was determined at several protein concentrations using time-lapse photography (26, 27). SDS disc gel electrophoresis. Polyacrylamide gels (10%) were prepared according to the procedure of Weber and Osborn (281. Samples were run at 5 mA/ gel, and gels were stained for protein with amido black dye. Absorption and circular dichroic spectra. Absorption spectra were obtained with a Cary Model 14 recording spectrophotometer equipped with O-O, 1 and O-l.0 absorbance slide wires using quartz sample cells of l- to lo-mm pathlength. Circular dichroism (CD) was determined with a Cary 61 spectropolarimeter using quartz sample cells of O.l- to lomm pathlength. Units of molar ellipticity, [0], are degrees per squared centimeter per decimole. The sample compartments of both instruments were continuously purged with N, to prevent oxidation of the cobalt enzymes. Sample cells were rendered metal free by soaking for 12 h in a 1:l solution of
508
ARTHUR
concentrated HNOs and H,SO, followed rinsing with metal-free distilled H,O.
by extensive
J. SYTKOWSKI
RESULTS
Enzyme Purification, Assessment of Homogeneity, and Molecular Weight
Isolation and purification of yeast alcohol dehydrogenase from fresh baker’s yeast by the method of Hvidt and Kagi (17) result in an enzyme with a specific activity of 400-425 U/mg after four recrystallizations. Further recrystallizations do not increase specific activity. The crystals obtained at this point are quite stable, and the loss of specific activity is less than 5% in 4 months when suspended in 20 mM sodium phosphate, 60% saturated in (NH4@04, pH 7.1, 4°C. Gel filtration of the enzyme through Sephadex G-200 increases the specific activity to 430-480 U/ mg. Disc gel electrophoresis of this enzyme in the presence of sodium dodecyl sulfate reveals a single band of apparent molecular weight of 37,000 & 3,000, slightly lower than the subunits of human and horse liver alcohol dehydrogenase (29). No other protein species is found. Sedimentation velocity experiments further confirm molecular weight homogeneity, demonstrating a single, narrow, symmetrical boundary. The apparent molecular weight (i&J was determined at several protein concentrations by ultracentrifuge time-lapse photography (see Materials and Methods). A plot of l/M,,, vs protein concentration is linear. Extrapolation to zero protein concentration yields a molecular weight of 149,000. The molar absorptivity (E) at 280 nm, assessed by gravimetric determination of the protein dry weight after dialysis of two 20-mg samples against H,O (see Materials and Methods), was found to be E = 1.83 x lo” M-’ cm2. (A!&% = 1.23 ? 0.01.) Metal Content of YADH
In YADH prepared as described above zinc is the only metal present in stoichiometrically significant amounts as determined by emission spectrography (24). Trace amounts of aluminum and magnesium are variably present while Ba, Ca, Cd, Co, Cr, Cu, Mn, MO, Ni, Pb, and Sn are not detected.
The Zn content of YADH was determined by atomic absorption, atomic fluorescence, and microwave emission spectrometry. Previous comparison of these three methods by analysis of several zinc metalloenzymes of known metal stoichiometry and homogeneity has demonstrated their accuracy, precision, and equivalence (14). Analyses of YADH prepared from fresh baker’s yeast under conditions which eliminate contamination by extraneous metal ions reveal 4.0 +- 0.3 gatom of zinc per mole of enzyme (Table I). No significant differences are found among the three analytical methods. YADH prepared in this laboratory from commercially dried yeast, the conditions and time of storage of which are unknown, has a slightly lower specific activity (400 U/mg) but similarly contains 4.2-4.3 g-atom of Zn/mol. Samples of commercially available enzyme consistently exhibit lower specific activities (260-400 U/mg) and, more important, their zinc contents are higher, ranging from 5.7 to 7.1 g-atom/m01 (Table I), suggesting contamination with adventitious metal atoms (see Discussion). There is no apparent correlation between the specific activities and zinc contents of these commercial enzyme samples. Efforts to obtain either a sample of BoehringerMannheim yeast from which enzyme could be prepared or details of their commercial preparative procedure were unsuccessful. Coenzyme Binding
The binding of NADH to YADH results in a slight hypsochromic shift of the NADH absorption spectrum. The new maximum is centered at 335 nm and exhibits a molar absorptivity, esX5,of 5880 M-’ cm-’ compared to es4,,= 6220 M-’ cm-’ for uncomplexed NADH (Fig. 1). This YADH *NADH complex is optically active with a negative circular dichroic extremum at 330 nm (Fig. 2, upper panel). A circular dichroism titration demonstrates an NADH/YADH binding stoichiometry of 4:l (Fig. 2, lower panel), identical to the zinc stoichiometry. These results are similar to those reported by Temler and Kagi (8). The 330~nm extremum has a maximum molar ellipticity, /&, of - 1.44 x 10” deg *cm2*dmol-’ , compared to that
PROPERTIES
OF YEAST
ALCOHOL TABLE
509
DEHYDROGENASE
I
SPECIFIC ENZYMATIC ACTIVITY AND ZINC CONTENT OF YEAST ALCOHOL DEHYDROGENASE FROM VARIOUS SOURCES Enzyme
Specific activity (LJ/mg)
source
Zinc content”
(g-atom/m01
Atomic absorption
Atomic fluorescenceb
of YADH) Microwave emission
Baker’s yeast preparations’ I(F)-12/74-l I(F)-l/75-2 I(F)-6/75-3 II(F)-8/75-4 III(F)-6/76-5 IV(F)-8/76-6 I(P-L&8/76-6 II(P-L)-8/76-8
460 430 450 480 460 470 400 400
4.0 3.9 4.3 3.7 4.0 4.0 4.2 4.3
4.1 4.1 4.2 3.8 3.9 4.0 4.4 4.2
3.9 4.1 4.1 3.9 3.9 4.0 4.2 4.4
Commercial YADH” B/M 7035436 B/M 7245437 B/M 7315537 B/M 1056138 B/M(X) 7235521 B/M(X) 7285321 S 84C-8570 S 114C-7830 S 45C-8241 W 35M 995
330 280 365 300 390 400 270 280 260 260
5.7 6.2 6.6 6.9 7.1 6.9 6.4 6.2 6.2 6.5
6.0 6.0 6.6 7.0 7.1 7.0 6.3 6.2 6.1 6.7
5.9 6.1 6.6 7.1 7.1 6.8 6.5 6.2 6.4 6.8
n All zinc contents shown are the averages of three to five measurements and replicate measurements all fall within a range of 25%. b Data kindly provided by Dr. Claude Veillon. c As prepared in this laboratory from baker’s yeast (see Materials and Methods). Roman numerals denote yeast batch. (F) = Federal Yeast Corp. (air dried); I(P-L) = P-L Biochemicals “active dried” (Lot No. 3850); II(P-L) = P-L Biochemicals “compressed baker’s” (Lot No. 3217). I(P-L) and II(P-L) were dried by the supplier. d B/M = Boehringer-Mannheim; S = Sigma Chemical Co.; W = Worthington Biochemicals. X denotes crystalline suspension; all other samples are lyophilized powders,
.-. : e w
4--
/’
‘\\ \
\
/I 2-- ’
-/\ 0
\\
//’
300
340 WAVELENGTH.
\\
\\
\\
\\
‘\
380‘ nm
FIG. 1. Effect of YADH binding on absorption spectrum of NADH. Free NADH, 340 pM c---j; NADH, 340 PM, in the presence of 190 FM YADH which equals 760 pM active site concentration (---). Spectrum of NADH-YADH complex determined vs 190 PM YADH blank. Buffer, 20 mM Na-phosphate, pH 7.5, 23°C.
of the NADH complex with horse liver alcohol dehydrogenase, OS85= -8.6 x lo4 deg. cm” *dmolk’, in which only 2 mol of coenzyme is bound (30). The ellipticities per mole of NADH of the YADH *NADH and LADH *NADH complexes are nearly identical, -3.6 x lo4 and -4.2 x 10” deg. cm’ *dmol-‘, respectively, suggesting similar modes of coenzyme binding in the two enzymes, consistent with, deductions made from nuclear magnetic resonance studies of NADH binding to YADH (31). The apparent equilibrium constant, K alw7 for NADH binding was assessed by CD titration of 2.3 PM YADH. A plot of l/ (0) vs l/[NADHh: is linear and reveals K aPP= 26 PM (Fig. 31, in close agreement with previously reported values (2, 8). The stoichiometry of NADH binding
510
ARTHUR 300 I
ri
350 I
J. SYTKOWSKI
I
4.5 for the peak and trough, respectively, in agreement with the values obtained by CD titration (see above). Conditions
OIY
4
12 NADH/YADH.
20
28
mols/mol
FIG. 2. Circular dichroism titration of [(YADH)Zn,l with NADH. Upper panel: The magnitude of the negative circular dichroic trough at 330 nm increases as NADH is added. Aliquots of 12.3 or 60 mM NADH (5-50 ~1) added sequentially. Lower panel: Extrapolation of titration data indicates binding stoichiometry of 4 mol of NADH/mol of YADH. Enzyme, 0.18 mM; 20 mM sodium phosphate, pH 7.5, 4°C. Units of molar ellipticity, 101, are degrees per squared centimeter per decimole.
of Zinc Exchange
The advantages of ‘j5Zn replacement of the stable zinc atom in the active site of a metalloenzyme have been clearly demonstrated (11). The determination of optimal conditions for Zn $ 65Zn exchange in YADH necessarily includes an examination of the effect of pH, which has been shown to be of importance both to metal exchange in enzymes (33) and to acid-assisted dechelation in model systems (34). Dialysis of YADH against 20 mM sodium phosphate in the absence of any added metal ions results in a time- and pH-dependent loss of activity at pH 4.6 and below (Fig. 5A). However, the addition of 047
s $
045 r-
0
20
60
40 EFFLUENT,
ml
FIG. 4. Stoichiometry of NADH binding to [(YADH)Zn,l by gel filtration method of Hummel and Dreyer (32). NADH, 680 PM; enzyme, 30 nmol applied in 50 ~1 of buffer; Na-phosphate, 20 mM, pH 7.5, 23°C; Bio-Gel P-4 (100-200 mesh), 1.5 x 26 cm, 0.8 ml/min, 500-~1 fractions; l-mm pathlength cuvette. FIG. 3. Double reciprocal plot of NADH circular dichroism titration of [(YADH)Zn,]. Titration of relatively low concentration of enzyme, 2.3 pM, with aliquots of 212 PM NADH. The intercept on the abscissa of l/(e) vs l/[NADH&,, is - l/K,,, constant of where Kapp = 26 PM, the dissociation enzyme-coenzyme complex (56).
was also examined by the gel filtration method of Hummel and Dreyer (32) (Fig. 4). Employing molar absorptivities at 340 nm of 5710 M-’ cm-’ for the complexed NADH and 6220 M-’ cm-’ for the free NADH (Fig. 11, calculation of the micromoles of NADH bound per micromole of YADH yields stoichiometries of 3.7 and
FIG. 5. (A) pa-dependent inactivation of YADH in the absence of added Zn. pH 5.0 (a), 4.6 (m), 4.4 (A), and 4.2 (r). (B) Zn protection of pH-dependent inactivation. pH 5.0 (0), 4.6 0, 4.4 (A), and 4.2 (v); 0.4 mM ZnSO1. Enzyme, 0.18 mM; 20 mM sodium at phosphate, 4°C. V,, control enzymatic activity zero time; V, observed activity at specified time.
PROPERTIES
OF YEAST
ALCOHOL
0.4 mM Zn2+ prevents this inactivation (Fig. 5B). These results are strikingly similar to those obtained by Drum (33) in his initial studies of zinc exchange in horse liver alcohol dehydrogenase and, hence, suggest possible conditions for metal exchange in YADH. Below pH 4.1, the enzyme rapidly denatures and is only 40% active after 6 h even in the presence of 0.4 mM Zn2+. The zinc content of YADH after dialysis against Zn2+ under these conditions is identical to that of the starting material, demonstrating that YADH does not acquire adventitious metal atoms when treated in this manner. The specific enzymatic activity of the resultant enzyme is 430-480 U/mg, identical to that exhibited by the homogeneous enzyme freshly prepared from baker’s yeast4 “VJinc Exchange
in YADH
The four zinc atoms of native [(YADH)Zn,] are replaced by ‘j”Zn under conditions of equilibrium dialysis at 4°C. Ten milliliters of [(YADH)Zn,l, 50 mg/ml, in 20 mM sodium phosphate, pH 7.5, is dialyzed against 1000 ml of 1 mM ssZnz+, 0.1 M sodium acetate, pH 4.20. This affords optimal yield of radiolabeled enzyme within a convenient time period. At various intervals aliquots of enzyme are removed from the dialysis bag and are analyzed for fi”Zn content. After equilibrium is reached, the exchange is terminated by changing the buffer to 20 mM sodium phosphate, pH 7.5. The sample is dialyzed for several hours against two additional changes of this buffer and centrifuged (6O,OOOg,30 min, 4°C) to remove precipitated material. The 4 g-atom of intrinsic zinc of the enzyme have exchanged fully after 8-9 h of dialysis against ““Zn (Fig. 6). During the exchange approximately 4 Enzyme obtained after only two (NH&SO, recrystallizations (see Materials and Methods) has a specific activity of 310-330 U/mg. Dialysis of such partially purified enzyme against Znz+ in this pH range also yields homogeneous enzyme of 430-480 U/mg containing 4 g-atom of Zn per mole after precipitated impurities are removed by centrifugation. Hence, this dialysis step can be used as a method for final purification eliminating two recrystallizations and gel filtration.
511
DEHYDROGENASE
30% of the enzyme precipitates, resulting in a final yield of 70%. As with LADH (33, 351, the rate of metal exchange in YADH is inversely related to protein concentration. Hence, this precipitation of enzyme during exchange results in progressively faster exchange. Thus, the apparent exchange data do not conform to simple firstorder kinetics. All four zinc atoms of YADH exchange identically in acetate or phosphate buffers, indicating that the four zinc atoms are equivalent. This is in marked contrast to horse liver alcohol dehydrogenase where the presence of these buffers differentiates between two noncatalytic and two catalytic zinc atoms (10, 11, 35). The zinc content, specific enzymatic activity, and coenzyme binding of “SZn-labeled yeast alcohol dehydrogenase, [(YADH)““Zn,l, are identical to those of the native enzyme (Table II), demonstrating that “;‘Zn has
FIG. 6. ““Zn exchange in YADH. ““Zn content by y emission spectrometry (0); 1 mM ‘“Zn’+, 20 mM sodium acetate, pH 4.2, 4°C. TABLE II ZINC CONTENT, SPECIFIC ENZYMATIC ACTIVITY, AND COENZYME BINDING OF I(YADH)““Zn,l [(YADHI““Zn,] preparations
BRL BRL BRL BRL
75-1 75-2 76-1 76-2
Total zinc content” (g-atom/ mol) Atomic absorption
y emission
4.1 4.0 4.2 3.9
4.2 3.9 4.2 4.0
Specitic activity W/ wd
NADHI YADHb (mol/ mol)
460 450 480 470
4.0 3.9 ND ND
(’ Total zinc content determined by both atomic absorption spectrometry and y emission spectrometry of “sZn. Results are 25%. * Circular dichroism titration; ND = not determined.
512
ARTHUR
indeed replaced the catalytically native zinc atoms. Cobalt Exchange
J. SYTKOWSKI
essential
in YADH
Substitution of cobalt for zinc in YADH is accomplished by dialysis of [(YADH) 65Zn41, 21 mg/ml, against a loo-fold volume excess of 0.1 M CoCI,, 20 mM sodium acetate, pH 4.50, 4”C, under 1 atm of oxygenfree N,. Two additional changes of the dialysate are employed to prevent back exchange of zinc. At specific intervals aliquots are removed from the dialysis sac, dialyzed free of extraneous metal atoms against 20 mM Tris-HCl, pH 7.5, 4”C, centrifuged to remove precipitated material, and analyzed for zinc, 6sZn, and cobalt. In the course of dialysis the zinc content of the enzyme decreases and cobalt content increases proportionally. Independent determinations of zinc content by atomic absorption spectrometry and y emission spectrometry are virtually identical, and at all times during exchange the total metal content of each sample, C (Zn + Co), is 4.0 + 0.2 g-atomlmol. Hence, the reliability of ““Zn loss as a precise measure of the rate, extent, and site specificity of cobalt replacement is demonstrated. When equilibrium is reached the exchange is stopped and free metal atoms are removed by dialysis of the enzyme against several changes of 20 mM Tris-HCl, pH 7.5, 4°C. The resultant enzyme, [(YADH)Co,], is green and contains 3.9 g-atom of cobalt and only 0.1-0.2 g-atom of zinc/mol. The specific enzymatic activity of the fully cobalt-substituted enzyme is 80 U/mg. This activity, while only 17% of that of the native enzyme, is much higher than can be accounted for by residual zinc (3-5% of that of the native enzyme). In addition, resubstitution of Zn into [ (YADH)Co,] results in a corresponding increase in activity, demonstrating that the reduced activity of [(YADH)CoJ is specifically due to replacement of the catalytically essential Zn atoms with Co and not to enzyme denaturation. Absorption
and Circular
Dichroic
Spectra
[ (YADH)CoJ exhibits absorption maxima at 340 and 627 nm with shoulders at
675-685 and 725-745 nm (Fig. 7). The molar absorptivities (6) are A,,,, (14,800), A627 u>7w, ~6,S+w (1,320), and h725-,45(680). The position and intensity of these maxima are strikingly similar to those exhibited by cobalt-substituted horse liver alcohol dehydrogenase, 1(LADH)Co$o,] (11). Associated with the 340-nm cobalt absorption maximum are two negative circular dichroic extrema centered at 332 and 388 nm and a shoulder at 310 nm with molar ellipticities, [el, of AsI0 G-11,900), A (-21,600), and A38X (-13,000) d$ *cm* *dmol-‘. In addition, a very weak ([Ol - 300-800) negative ellipticity is present at 550-700 nm (not shown) corresponding to the absorption maxima in this region (Fig. 8). DISCUSSION
Over 20 years ago YADH was shown to contain 4 g-atom of catalytically essential zinc per mole (1) and to bind 4 mol of coenzyme (2). Subsequently, Wallenfels and Sund (3, 4) reported stoichiometries of 5:l for metal content and coenzyme binding. Later, several reports confirmed the metal content of 4 g-atom/m01 (6, 7, 15, 36). However, recently both the coenzyme 2 3 PQ * Y
m x 0 1 L
2
0lud
- 1 350
-
0
450
600
700
WAVELENGTH.nm
FIG. 7. Absorption spectrum Tris-HCl, 20 mM, pH 7.5, 4°C.
L-&
-d
of
[(YADH)Co41.
+ 400
W&VELENGTH,
nm
dichroic spectrum of FIG. 8. Circular [(YADH)Co41. Tris-HCl, 20 mM, pH 7.5, 4°C. Units of molar ellipticity, [Ol, are degrees per squared centimeter per decimole.
PROPERTIES
OF YEAST
ALCOHOL
binding and zinc stoichiometries have again come into question. The NADH/ YADH ratio of 3:l reported by Dickinson (37) and by Sanderson and Weiner (38) has been revised downward to 2:l (7), similar to the ratio of 1.751 reported by Leskovac and Pavkov-Pericin (39). In addition, Dickinson’s earlier zinc stoichiometry of 4.3-5.0 g-atom/m01 (7) has also been revised to 7.6 g-atom/m01 for a “relatively impure” enzyme preparation (40), a value similar to the 7.1-7.4 g-atom/m01 reported by Klinman and Welsh (16). These findings of a higher zinc content were subsequent to enunciation of the hypothesis of Branden et al. (13), based on the partial sequence homologies between YADH and LADH, that, as in LADH, two classes of zinc atoms might exist in YADH. Hence, YADH might contain not 4 but 8 g-atom of Zn. This prompted a reinvestigation of the Zn content of homogeneous YADH by means of three highly sensitive and accurate analytical methods, which have confirmed that the intrinsic zinc content is 4 g-atom/m01 (14). YADH readily binds adventitious metal atoms, as was well documented by Wallenfels and Sund (3, 4), Redetzki and Nowinski (41), and Hoch and Vallee (42). The metal stoichiometries of YADH preparations obtained from several commercial sources are variable and in excess of 4 gatom of Zn/mol (Table I). This excess almost certainly reflects adventitiously bound zinc acquired in the course of enzyme isolation. The lack of detailed information regarding the preparative procedures used in these instances precludes a definitive statement regarding the source of contamination (vide infra). Wallenfels and Sund (3, 4) and Sund (5) reduced the excess zinc content of YADH from a total of 35 to 4.7 g-atom per mole by dialysis against EDTA. They suggested that the chemical environment of the enzyme during isolation and even the properties of the yeast culture itself might result in binding of adventitious zinc atoms. Hoch and Vallee (42) showed that the zinc content of a preparation with 5.2 g-atom of zinc per mole was reduced to 4.0 g-atom by dialysis at pH 6.0 while both specific enzymatic activity and the number of
DEHYDROGENASE
513
titrable sulfhydryl groups actually increased, consistent with the demonstrated inhibition of YADH activity by adventitious metal atoms and reactivation upon their removal by chelating agents (5, 41). Recent studies confirm that EDTA removes excess zinc from YADH (14, 16) although under certain conditions of dialysis the enzyme is apparently unstable and loses activity (16). The problem of adventitious metal binding is not unique to YADH, of course, and is an important consideration in the study of all metalloenzymes (43). It is critical that investigations of such enzymes be carried out on well-characterized homogeneous material and that particular attention be given to the prevention of metal contamination. Metal contamination can arise from many sources. Buffer contamination may result from impure reagents or water (including acids or bases used to adjust pH), from pH electrodes, from containers used to prepare and store the buffers, and even from airborne sources. Consequently, solutions must be prepared from materials known to be free of metals or these extraneous metals must be removed either by solvent extraction with a chelating agent or by passage through an ion exchange resin. Even then, it cannot be assumed that the solutions so treated are necessarily rendered free of contaminating metals, since, for example, treatment with ion exchange resins may remove one contaminant and contribute another. Thus, the absence of contamination must be verified by suitable metal analysis. Gel filtration and ion exchange media, and associated equipment used for enzyme purification, must be similarly free from metals. In the present isolation and purification of YADH strict attention was paid to the elimination of all sources of metal contamination. The resultant enzyme is homogeneous, of high specific activity, and contains 4 g-atom of catalytically essential, intrinsically bound Zn per mole of enzyme. This stoichiometry is identical to that found for coenzyme binding and indicates one Zn atom and one coenzyme binding site per subunit. The detailed X-ray crystallographic
514
ARTHUR
J. SYTKOWSKI
analysis of horse liver alcohol dehydrogenase (13) has confirmed the conclusions of previous studies on the nature of the zinc atoms of that enzyme (9, 35) and has further delineated knowledge regarding coordination of its metal atoms. The partial homologies in the amino acid sequences of horse LADH and YADH described previously (12) led to the suggestion that the tetrameric yeast enzyme might contain two firmly bound Zn atoms per subunit, i.e., eight per tetramer, analogous to the catalytic and noncatalytic Zn atoms of the dimeric horse enzyme, or, alternatively, that the cysteinyl residues in the yeast enzyme which are homologous to ligands of the noncatalytic Zn atoms in the horse enzyme may form S-S bonds (13). The results of the present study show that there are only four firmly bound catalytically essential zinc atoms per tetramer in YADH. There is no evidence for a second, firmly bound structural zinc atom per subunit in this enzyme. Apparently the tertiary structure of the region in YADH containing these homologous cysteinyl residues does not permit the firm binding of an additional zinc atom. Importantly, comparison of the now completed amino acid sequence of YADH (44) to that of LADH reveals some striking differences between the two enzymes, especially in that region (residues 90-149) which binds the noncatalytic Zn atom in LADH. In this region only “random similarities” exist between LADH and YADH strongly suggesting that potential Zn ligands in YADH “need not be conserved in space although strictly homologous in primary structure” (45). The replacement of the zinc atoms of native enzymes by other metals with more favorable spectral properties has greatly facilitated the elucidation of the role of metal atoms in the mechanism of action of metalloenzymes (43). In this regard, the chromophoric and paramagnetic cobalt atom has been of particular value in the investigation of carboxypeptidase (461, carbonic anhydrase (47), alkaline phosphatase (48, 491, thermolysin GO), horse LADH (10, 11, 51), leucine aminopeptidase (521, and other enzymes (43). In contrast
to most other metalloenzymes, it has thus far not been possible to prepare stable apo-LADH by exposure to chelating agents, thus rendering it impossible to study the restoration of activity by direct addition of zinc or other metal atoms. Therefore, methods to exchange metals by equilibrium dialysis were devised to replace the native zinc atoms in LADH (33, 35, 53). Similarly, it has not been possible to remove the catalytically essential zinc atoms from YADH without apparently irreversible denaturation of the enzyme. In the present study, the methodological principles developed for metal exchange in horse LADH (33) have been applied to studies of the yeast enzyme, and the conditions for replacement of the native zinc atoms of this enzyme with ““Zn and subsequently with Co have been derived. It is essential that metal exchange be performed with enzyme of high purity and specific activity and of known, uniform metal stoichiometry, criteria which demanded rigorous investigation of the methods for the isolation, purification, and characterization of YADH prior to undertaking exchange studies. The enzyme prepared and purified from fresh yeast as described here meets these criteria. Under all conditions investigated the exchange properties of the four Zn atoms in YADH were found to be the same. All four atoms exchange identically in either the phosphate or acetate buffers used, in striking contrast to LADH, where the differences in exchange characteristics distinguish between the catalytic and noncatalytic Zn atoms (11, 35). This suggests that the four Zn atoms in YADH are equivalent, and it may then be presumed that they are located in identical environments within the enzyme, i.e., one zinc atom at each active site. Importantly, the exchange is pH dependent over a very critical, narrow range (Fig. 5), approximately 1 pH unit lower than that of the horse enzyme, and is extremely sensitive to slight deviations in hydrogen ion concentration. [ (YADHjG5Zn,l has the same specific activity metal content and coenzyme binding properties as the native en-
PROPERTIES
OF YEAST
ALCOHOL
zyme. Using this radiolabeled enzyme, it is now possible to substitute other metal atoms, such as cobalt, for zinc in YADH and to be certain of the site specificity and extent of such substitution, as shown for LADH (11). Since YADH readily binds adventitious metal atoms (vide supra), the location of any “replacement” atoms in the enzyme is open to serious question unless the simultaneous loss of the intrinsic, catalytically essential zinc atoms is unambiguously demonstrated by such quantitative, site-specific means. Previously, YADH containing metal atoms other than Zn has been obtained by isolation of the enzyme from yeast grown in medium rich in the respective metal. Curdel and Iwatsubo (15) prepared a green enzyme with approximately 1.2 g-atom of Co and 2.3 g-atom of Zn/mol. The absorption spectrum of this enzyme exhibited broad maxima at 620, 670, and 710 nm qualitatively similar to those obtained in the present study. Unfortunately, a specific activity for this enzyme was not reported. Another example of metal replacement in YADH by biosynthetic means is the isolation of a manganese YADH by Coleman and Weiner (6). This enzyme contained 3.7 * 0.6 g-atom of Mn/mol and no detectable Zn. The enzyme was far less stable than the corresponding Zn enzyme and, hence, it was necessary to store it in the presence of 20% sucrose and 0.1% mercaptoethanol. Also, 1 mM EDTA inactivated the Mn enzyme but not the Zn enzyme. Recently, a series of “hybrid enzymes” containing 1.0-2.9 g-atom of Co and 3.8-5.6 g-atom of Zn per mole of YADH have been prepared by dialysis of enzyme containing 7.0 g-atom of Zn against Co”+ (16). The specific locations of these replacement metal atoms in the enzyme have not yet been ascertained. The present study is the first report of the preparation of cobalt-substituted yeast alcohol dehydrogenase in which the site specificity and extent of the cobalt substitution are precisely delineated. The resultant fully substituted enzyme, [(YADH)Co,], contains 3.9 g-atom of Co and only 0.1-0.2 g-atom of Zn per mole. The data indicate that in this enzyme Co
DEHYDROGENASE
515
has specifically replaced the four intrinsic, catalytically essential Zn atoms. By analogy with the two catalytic, active site metal atoms of horse liver alcohol dehydrogenase, a dimer, the four intrinsic metal atoms of YADH, a tetramer, might be presumed also to lie in the active sites of the enzyme, one per subunit. Hence, the Co atoms of [(YADH)Co,l would serve to reflect events occurring at this locus. This is supported by the change in specific enzymatic activity which ensues from this metal replacement, similar to that observed upon Co replacement of the catalytic but not the noncatalytic Zn atoms of horse LADH (11). Moreover, the absorption spectrum of [(YADH)Co,l strongly suggests similarities in coordination of Co in this enzyme with the cobalt atoms of substituted enzyme, the horse l(LADH)Co2Co,l. The 340-nm absorption maximum of [(YADH)Co,l exhibits a molar absorptiviity (e) of 15,800, i.e., 395O/Co atom, virtually identical to 3750/catalytic Co atom observed in [ (LADH)Co,Co,]. The position and intensity of this maximum likely represent charge transfer between Co and sulfhydryl ligands (54, 55) which have been demonstrated to coordinate the active site metal in LADH (13) and which amino acid sequence homology suggests are present in YADH (44, 45). The visible absorption maxima of l(YADH)Co,] and l(LADH)Co,Co,l are also similar in position and intensity and are consistent with distorted tetrahedral coordination geometries in both enzymes (55). In contrast, the circular dichroic spectrum of [(YADH)Co,l is strikingly different from that of [(LADH)Co,Co,l (A. J. Sytkowski and B. L. Vallee, in preparation). Although a detailed interpretation of these CD spectral characteristics is not possible at present, they clearly are indicative of the asymmetry inherent in the coordination complex of the catalytically essential cobalt atom. These data establish the physicochemical, analytical, and enzymatic properties of highly purified YADH as well as the conditions for ““Zn and cobalt exchange. This knowledge now permits the detailed investigation of the role of the catalyti-
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tally essential metal atoms in the enzyme. Taken together, these methods illustrate a general approach to the elucidation of the functional and structural roles of metal atoms in metalloenzymes (57). ACKNOWLEDGMENTS The author wishes to thank Dr. J. L. Bethune and Dr. W. Dafeldecker for valuable assistance and discussions of the analytical ultracentrifuge data, Dr. Gerald M. Alter for advice on the circular dichroism titrations, and Dr. Claude Veillon for his participation in the early phase of this work (14). REFERENCES 1. VALLEE, B. L., AND HCICH, F. L. (1955) PFOC. Nat. Acad. Sci. USA 41, 327-338. 2. HAYES, J. E., JR., AND VELICK, S. F. (1954) J. Biol. Chem. 207, 225-244. 3. WALLENFELS, K., AND SUND, H. (1957) Biochem. z. 329, 31-40. 4. WALLENFELS, K., AND SUND, H. (1957) Biochem. z. 329, 59-74. 5. SUND, H. (1957) Ph.D. thesis, Hamburg. 6. COLEMAN, P. L., AND WEINER, H. (1973) Biochemistry 12, 3466-3472. 7. DICKINSON, F. M. (1974) EUF. J. Biochem. 41, 31-36. 8. TEMLER, R. S., AND K;~GI, J. H. R. (1973) Abstracts, IXth International Congress on Biochemistry, Abstract 2r4, p. 109. 9. DRUM, D. E., AND VALLEE, B. L. (1970) Biochemistry 9, 4078-4086. 10. SYTKOWSKI, A. J., AND VALLEE, B. L. (1975) Biochem. Biophys. Res. Commun. 67, 14881493. 11. SYTKOWSKI, A. J., AND VALLEE, B. L. (1976) PFOC. Nat. Acad. Sci. USA 73, 344-348. 12. JBRNVALL, H. (1973) Proc. Nat. Acad. Sci. USA 70, 2295-2298. 13. BRKND~N, C.-I., JGRNVALL, H., EKLUND, H., AND FURUGREN, B. (1975) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 11, Part A, pp. 103-190, Academic Press, New York. 14. VEILLON, C., AND SYTKOWSKI, A. J. (1975) Biothem. Biophys. Res. Commun. 67, 1494-1500. 15. CURDEL, A., AND IWATSUBO, M. (1968) FEBS Lett. 1, 133-136. 16. KLINMAN, J. P., AND WELSH, K. (1976)Biochem. Biophys. Res. Commun. 70, 878-884. 17. HVIDT, A., AND K;~GI, J. H. R. (1963) Compt. Rend. Trav. Lab. Carlsberg 33, 497-534. 18. THIEFLS, R. (1957) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 5, pp. 273-335, Wiley-Interscience, New York. 19. ALVAREZ, R. A., PAULSEN, P. J., AND KELLEHER, D. E. (1969) Anal. Chem. 41, 955-958. 20. FUWA, K., PULIDO, P., MCKAY, R., AND VALLEE,
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