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Some physical and chemical properties of crystalline α-glycerophosphate dehydrogenase
.4RCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
76,
125-141 (1958)
Some Physical and Chemical Properties of Crystalline ~Glycerophosphate Dehydrogenase’ Ho Lee Young and Nello Pace From the Department
of Physiology, School of Medicine, Rerkeley, California Received
September
Cniversit!l
of California,
19, 1957
INTRODUCTION
a-Glycerophosphate dehydrogenase act,ivity was first observed by Meyerhof (1) in 1919, and since that time two distinct a-glycerophosphate dehydrogenases have been isolated from mammalian muscle. The first, which is apparently associated with the cell particulates and which does not require the presence of diphosphopyridine nucleotide (DPN) for its action, was isolated and studied thoroughly by Green (2) an 1936. Von Euler and co-workers (3) in 1937 isolated from rabbit muscle another cY-glycerophosphate dehydrogensse which does require DPN as a hydrogen acceptor, and which has been termed the “soluble” Lu-glycerophosphate dehydrogenase, t’o distinguish it from the “particulate” a-glycerophosphate dehydrogenase of Green. It is t’he soluble cr-glycerophosphate dehydrogenase with which t,he present study is concerned. The soluble enzyme has been crystallized previously by Baranowski (4) and Beisenherz et al. (5); however, information on the physical properties of crystalline soluble a-glycerophosphate dehydrogenase is sparse, and the present paper describes several additional physical properties of the enzyme. Chemical properties of soluble a-glycerophosphate dehydrogenase have also been studied previously by several workers. The equilibrium constant for the oxidation of a-glycerophosphate has been measured by von Euler et al. (3, 6), Green et al. (7), Baranowski (4), and Burton and Wilson (8). The specificity of the enzyme and the Michaelis constant for t,he oxidation of Lu-glycerophosphate t’o dihydroxyacetone phosphat,e 1 These studies were supported 222(31) between the Office of Naval
under Contracts K7onr-295(04) and NonrResearch and t,he University of California. 125
126
YOUNG
AND
PACE
has been determined by Green et al. (7). The present study represents an examination of these and other chemical properties of the crystalline enzyme. MATERIAL
AND
METHODS
Reagents 1. D, L-or-Glycerophosphate and L-a-Glycerophosphate. Sodium D,L-a-glycerophosphate was made according to the method described by King and Pyman (9). The barium L-u-glycerophosphate used was kindly donated by Dr. E. Baer of the University of Toronto. The latter salt was dissolved directly in distilled water for low concentrations, but for high concentrations it was converted to the potassium salt immediately before it was used in an experiment. 2. Dihydroxyacetone Phosphate. The calcium salt was prepared enzymically by the method of Meyerhof and Lohmann (10) and was stored under vacuum in the cold for not longer than 2 weeks. The amount of dihydroxyacetone phosphate was determined immediately before use by the iodine oxidation and alkali-labile phosphate determination as described by Stumpf (11). The freshly prepared dihydroxyacetone phosphate contained an average of 90% dihydroxyacetone phosphate, a maximum of 0.5yo inorganic phosphate, and approximately 10% other phosphate, which probably consisted mostly of hexose diphosphate and glyceraldehyde phosphate. None of these impurities interferes with the assay of the crystalline enzyme. When low concentrations of dihydroxyacetone phosphate were required, the calcium salt was used directly; however, when high concentrations were needed, it was converted to the potassium salt. 3. Other Chemicals. Double glass-distilled water was used. Ca hexose diphosphate (Schwarz Laboratories, Inc.), DPN, and DPNH (Sigma Chemical Co.) and bovine albumin (Armour Laboratories) were also used.
Enzyme Assay Methods 1. Assay Method I (Oxidation of DPNH). This method is based on the oxidation of DPNH by dihydroxyacetone phosphate in the presence of cu-glycerophosphate dehydrogenase to form a-glycerophosphate and DPN, as described by Baranowski (4). The amount of DPNH oxidized and the rate of the oxidation were determined by measuring the change in light absorption at 340 rnr, using a Beckman model DU spectrophotometer. Readings of the spectrophotometer were made every 30 sec. until the reaction was essentially complete, usually u-ithin 5 min. after the start of the reaction. 2. Assay Method II (Reduction of DPN). This assay was based on the reverse of the chemical reaction described in the preceding section. The rate of appearance of DPNH in the reaction mixture was measured by following the change in light absorption at 340 mp.
Protein Determination The concentration biuret method (12).
of protein
in the enzyme solutions
was determined
by Kirk’s
a-GLYCE~OPHOSPH.4TE DEHYDHOGES.4SE PIZOPEHTIES
I’Ic:. I. wGlyc:erophosphate
dehydrogenase
crystals
from
rabbit
muscle
127
(2&5X).
RESULTS
C‘r+stallixation
of cY-Glycerophosphate Dehydrogenase
The prcparut~ion of myogen X crystals from rabbit muscle was carried out by the met’hod described by Baranowski and Kiederland (13) wit,h slight modification, to separate the sediment,ed protein by wntrifugntion inst,ead of by filtration. Preparation of crystalline a-glycerophosphate dehydrogenase was performed as described by Baranowski (4) ; however, the crystals obtained in this study were rectangular plates, as shown in Fig. 1, lvhilr those prepared by Buranowski were rhombic plates. The crystalline errzyme was stable for several weeks when it’ was stored as a suspension in npproximately 50 7%(KH,)$30., in the cold. Aldolase activity could trot’ be det ected by thr method of Baranowski and Sicderland (I:<). Phosphatasr acti\4ty lilwwisc was not detectable by the technique of Da\-ier (II).
The ultjraviolct absorption spectrum from 210 to 300 mp ~w:: generally mow similar to that measured by Baranowski (4) thalr to that of Beisrw hcrz cl rtl. (15), but exhibited some differences front Iwth. ‘l’hc present
128
YOUNG
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preparation had a somewhat sharper absorption peak around 280 rnp, and, in general, the extinction coefficients at all wavelengths were from 2 to 20% lower than those reported by Baranowski. Beisenherz et al. (15) found that the maximal absorption of the enzyme was at 270 rnp, whereas the present preparation showed a maximal absorption at 278 rnp. At 270 rnp the extinction coefficient of t,he present preparation was about 20% lower than that of the Beisenherz preparation. Sedimentation Coej’kient a-Glycerophosphate dehydrogenase crystals were filtered from their (NHh)$304 suspension by suction through Whatman No. 50 filter paper. Samples of the relatively dry crystals were dissolved in a small amount of water and dialyzed against 0.5 M (NHJzS042 for 18 hr. in the cold. The cr-glycerophosphate dehydrogenase solutions were centrifuged in a Spinco model E ultracenbrifuge analyt’ical rotor at 59,780 r.p.m., an average centrifugal field of 260,000 X g, with refrigeration. The sedimentation coefficient, under the standard condition of pure water at 2O”C., szo, -a., was calculated by the Svedberg and Katsurai (16a) equations. An ultracentrifuge pattern of a-glycerophosphate dehydrogenase is shown in Fig. 2. The sedimentation coefficients calculated from the upper and lower patterns are 6.4 X IO-l3 sec. and 6.5 X lo-l3 sec., respectively. Sedimentation coefficients were det’ermined for several protein concentrations of two separate enzyme preparations, and the values of ~20,w were plotted against the protein concentrations for each preparation. The szn, ,” value was only little affected by change in protein concentration in the range studied from 0.2 mg./ml. to 10 mg./ml. The zeroprotein-concentration extrapolated values of szo, w were found to be 6.4 X lo-l3 sec. and 6.6 X lo-l3 sec., respectively. The difference was regarded as not significant within the limits of accuracy of the method; hence the mean value of 6.5 X lo-l3 sec. was taken to represent the sedimentation coefficient of a-glycerophosphate dehydrogennse in water at 2O’C. * Ammonium sulfate, 0.5 84, instead of KC1 was used to prevent the electrostatic effect on particle sedimentation. The wglycerophosphate dehydrogenawe precipitated out and did not redissolve in water when it was suspended in 0.5 M KCl. Two possible explanations for this behavior may be mentioned: either the ionic strengt,h of 0.5 M KC1 may have been too low, or the enzyme was denatured by either or both the potassium and chloride ions.
FIG. 2. Ultracentrifuge patterns of ru-glycerophosphate dehydrogenase as a function of time. The dialyzed cnzymc solution in the upper patt,ern contained 2.7 mg. protein/ml. of 0.5 M (NHI)&304 , and the dialyzed solution in the lower pattern contained 0.51 mg. protein/ml. of 0.5 M (NHa)280a , both at, pH 5.6. The rotor speed was 5!),780 r.p.m. and the Itar angle was 60”. The times of each frame were 0, 8, 16, 24, and 32 min. after rcachillg full sl~cctl. ‘I’hc initial 1enipcraturr ~vas 6.0”( ‘_ and t hcl final tcmpcraturc kvas 8.4”(‘.
s
3 ? Fs
130
YOUNG
AND
spparent
PACE
SpeciJic Volume
The apparent specific volume, 7, determination was carried out by weighing the solvent, 1 M (NHI) $04 , alone and then weighing an a-glycerophosphate dehydrogenase solution of 0.0040 g./cc. in a pycnometer containing 0.9906 ml. at 24°C. The enzyme solution was previously dialyzed against 1 M (NHI)$04 . The apparent specific volume was calculated by means of the equation derived by Kraemer (16b). The weight of the dialyzed 0.0040 g./cc. enzyme solution, m, was 0.1063 g. The weight fraction of the dialyzed enzyme solution (~1 = gv/m) was 0.0037, the weight fraction of the solvent (WZ = 1 - ~1) was 0.9963, and the specific volume of solvent (V,) was 0.9328 ct./g. The results are shown in Table I, and the mean apparent specific volume of a-glycerophosphate dehydrogenase from five determinations was found to be 0.75 ct./g. TABLE
I
Mean of Five Deternlinations
of the Apparent Specific Volamz of a-Glycerophosphate Dehydrogenase A pycnometer containing 0.09906 ml. (v) at 24°C. was used.
Determination
1 2 3 4 5
Weight
of 1 M (NH&SOa
(mo)
Specific solution
0.1060 0.1060 0.1060 0.1061 0.1060
volume
of
(V = v/m)
Apparent specific volume of a-glycerophosp&te dehydrogenase (V)
0.9319 0.9319 0.9328 0.9319 0.9328 Standard
Mean deviation f
0.70 0.70 0.95 0.70 0.70 0.75 0.11
viscosity The enzyme solutions used for viscosity measurements were prepared in the same way as the one used for the apparent specific volume study. The flow time for the solvent and the enzyme solutions was determined five times in a microviscometer of the Ostwald type and expressed as an average for each solution, as shown in Table II. The relative viscosity, qr , and the ratio of specific viscosity, vsD, to the volume fraction of the solute were calculated by the methods described by Alexander and Johnson (17). The results are shown in Table II. Mean values for as,/+ of 8.6 and 10.0 were found for the two concentrations of a-glycerophosphate dehydrogenase. Because of the limited amount of crystalline en-
a-CLYCEROPHOSPHATE
DEHYDROGENASE
131
PROPERTIES
Mean oj” Five Determinations of the IZatio of SpeciJic Viscositll to Volume Fraction (Q,/+) at ~5.5%‘. 0.407,
Determindtion
solution
Flow time of 1 ‘K (hTHh)nS0~ !I?)
Flow time of en7.yme solution (11)
0.807~ solution
dchydrogenase
of a-glycero hosphate in 1 M ( ff H&SOa
dehydrogenase
Relative viscosity enzyme solution (WY
of
Ratio of specific viscosity to volume fraction (?.P/Sbi”
Flow time of enzyme solution ill)
SK. 1
2 3 4 5 Mean s. D.
of a-glycerophosphate in1 .44 (KH~)zSOI Relative viscosity of enzyme solution (4ri
Ratio of specific viscosity to volume fraction f?SP/V+)
1.060 1.060 1.060 1.060 1.059 1.060 fO.0005
10.0 10.0 10.0 10.0 0.8 10.0 fO.10
SM.
71.43 il.35 71.33 il.45 71.42 71.40 f0.053
73.18 73.16 73.18 73.16 73.1-I 73.16 f0.014
1.026 1.026 1.026 1.026 1.025 1.026 fO.0005
I’ 7r = pltl/pnt2 , where deusity of 1 112 (KH,)&Wd h ll.Y,/@ = or -
8.7 X.7 8.7 8.7 8.3 8.6 f0.17
of enzyme
solution,
75.58 75.59 75.58 i5.48 75.57 75.56 fO.045
p, . was l.Oi3,
and density
, p2 , \vas 1.072. 1.
4
zyme available, only these two concentrations were studied. However, the value of qlsP/+ at infinite dilution was obtained by extrapolation to $ = 0 from the two values obtained, when qs,,/$ was plotted against 4, and was found to be 7.2. This is probably a reasonable approximation of the true ratio. From the viscosity data a frictional ratio, f/j” , of 1.4 was obtained from the Oncley contour diagrams (18), using the value of qsP/4 of 7.2. The molecular weight, M, could then be computed by the following equnt8ion : M2’3 = (f/f,,) (Gav,sN),‘(l
- VP,) (3v/47rN)1’3
where qWis the viscosity of water; N is the Avogadro number, 6.0235 X 10z3/mole; and pa is the density of 0.5 M (NHd)zSO., . The molecular weight by this calculation was found t,o be 173,000 g.,/mole. Stability
of the Enzyme
Both thermal stability and the pH stability were examined. The enzyme was found to be relatively thermolabile, 53 % of it,s activity being lost by heating to 55°C. for 1 min., and nearly all activity being lost at
132
YOUNG
AND
PACE
60°C. However, there was no appreciable loss of activity when it was kept at room temperature, 22”C., for as long as 30 min. The pH stability was studied at two different temperatures, 1.5 and 28”C., using barbital buffer solutions (19). All the solutions were kept at the particular pH and temperature for 2 hr. before testing their activity. The results are shown in Fig. 3, and the highest rate at pH 6 was taken as 100 %. The enzyme was most stable around pH 5.7, both in the cold and at 28°C. The protein was precipitated by extreme acid or alkali concentrations at both temperatures, and the precipitated protein was no longer soluble in water. Another experiment was done to test the stability of the enzyme. A solution containing 1 mg. protein/ml. in approximately 0.2 N ammonium sulfate at pH 5.8 was kept between 0 and 4°C. The activity of the enzyme was measured at intervals during a 2-week period. It was found that there was no change in enzyme activity during this time. Curves
pH-Activity
The effect of pH on the reduction of dihydroxyacetone phosphate is shown in Fig. 4, and the highest rate of the reaction was found at ap-
----k-g
4
PH
6
6
FIG. 3. Effect of pH on the stability of two portions of an a-glycerophosphate dehydrogenaae solution: one kept at 1.5”C. and the other kept at 28°C.
WGLYCEROPHOSPHATE
j.; -
65
DEHYDROGENASE
PROPERTIES
,
,
;
70
75
80
133
8
PH
FIG. 4. The effect of pH on the rate of reduction of dihydroxyacetone phosphate in the presence of a-glycerophosphate dehydrogenase. Dihydroxyacetone phosphate, 2.7 X lo-” M, WE used as the substrate.
pH 7.5. The effect of pH on the oxidation of L( -))cr-glycerois shown in Fig. 5, and the highest rate of oxidation occurred at about pH 10.2. For these curves 0.2 M phosphate buffer was used to obt,ain the pH values between 6.7 and 8.6, and 0.03 M glycine buffer was used in the pH range from 1.5 to 3.5 and from 8.5 to 11.5. proximately phosphate
Michaelis Constant In the present instance, where five components are involved, application of the Lineweaver and Burk treatment (20) to the kinetic data obtained by varying the concentration of one component at a time leads only to approximate values for V, and K, for each component, and hence complete determination of the constants cannot be made (21). The values obtained, however, are useful in establishing gross characteristics of the reaction involved. The relationship between the rate of reaction and the concentration of different substrates is shown in Figs. 6 and 7, while the relationship between the rate of reaction and DPN concentraCon is shown in Fig. 8.
134
YOUNG
AND
PACE
FIG. 5. The effect of pH on the rate of oxidation of L-ol-glycerophosphate in the presence of a-glycerophosphate dehydrogenase. a-Glycerophosphate dehydrogenase, 3.5 X 10-S M, was used as the substrate.
The maximal velocity for each reaction, obtained from Figs. 6-8, and the calculated Michaelis constants are given in Table III. Table III shows that the Michaelis constant, K, , for the oxidation of a-glycerophosphate is 1.1 X 10e4 M. This value is low in comparison with the one reported by Green (7) of 1.25 X 10e3 M. In the Lineweaver-Burk plot of reaction velocity against DPNH concentration, all points were fairly scattered. A reliable estimated Michaelis constant for DPNH could not be obtained; however, its concentration effect appeared to be much less. Table IV shows the marked dependence on temperature of the rate of reduction of dihydroxyacetone phosphate, and hence of the value for the turnover number for cu-glycerophosphate dehydrogenase. Thus in any comparison of values for turnover number obtained by different investigators, the substrate concentration, the pH, and the temperature must be taken into account. Baranowski (4) has reported a turnover number of 45,800 moles sub-
a-GLYCEX~OPHOSPHA!l’E
DEHYDROGENASE
I T
10’
PROPERTIES
135
ml. /mole
FIG. 6. Lineweaver-Rurk plot of reaction velocity against r,-a-glgcerophosphate concentratjion at, pH 7.00. The reaction mixture, totaling 3 ml., contained 1 ml. of 2.44 X 10F3 .?I Dl”?;T (in 0.2 M phosphate buffer at pH 7.00), I ml. of sodium or-glycerophosphste solution ranging from 1.2 X 10-d M to 5.7 X UP M, 0.9 ml. of distilled water, and 0.1 ml. of enzyme solution containing 140 pg. protein/ml. The temperature at which the reactions were measured was 23.3”C.
strate/miu./lOO,OOO g. protein for a-glycerophosphatje dehydrogenase, using 0.28 M dihydroxyacetone phosphat’e as t’he sub&ate at pH 7.0 nlld 22”C., and Beisenherz et al. (15) reported an estimated turnovel number of 51,900 at 25°C. From t,he Michaelis constant of 4.6 X 10m4&l for the reduction of dihydroxyacetone phosphate shown in Table III, a turnover number of 84,400 moles/min./mole of enzyme at pH 7.0 and 22°C. may be computed. The DPNH used in determining K, for dihydroxyaretone phosphate was about double the amount used by Baranomski for turnover study. Thus t,he turnover number for the crystalline enzyme preparutSion used in the present study at, the same pH would appear to he substant,ially greater than that obtained by either Barnnowski or Beisenherz et al. ; however, such a comparison may not be valid because of the widely differing sub&rate concentrabions used in the two studies.
136
YOUNG
AND
PACE
62 E . ri :: 2 E
$ -I>
540
/
32,000
;Pp 0
I
2
I 5
IO’
I 4 ml./mols
I 6
a
O/ a/ 0
6-
I
0 0
0.5
( r
I I.0
I 1.5
, 2.0
IO' ml. /mole
7. Lineweaver-Burk plot of reaction velocity against dihydroxyacetone phosphate concentration. (a) Upper graph: The reaction mixture contained 1 ml. of 6.15 X 1O-4 M DPNH (in 0.2 M phosphate buffer at pH 7.00), 1 ml. of sodium dihydroxyacetone phosphate solution ranging from 1.0 X 10e6 M to 3.0 X lO+ M, 0.9 ml. of distilled wat.er, and 0.1 ml. of enzyme solution containing 200 pg. protein/ml., at 23°C. (b) Lower graph: The reaction mixture contained 1 ml. of 7.35 X lo-” M DPNH (in 0.2 M phosphate buffer at pH 7.42), 1. ml. of dihydroxyacetone phosphate solution, ranging from 1 .O X 10e6 M to 3.0 X 10e3 M, 0.9 ml. of distilled water, and 0.1 ml. of enzyme solution containing 20 pg./ml., at 24°C. FIG.
Equilibrium
Constant
The equilibrium constant for the reduction of dihydroxyacetone phosphate has been measured by von Euler (3, 6), Green (7), Baranowski (4), and Burton and Wilson (8). However, their results are not consistent. In the present work the equilibrium constant was measured by assay method II at 24.5%. and was computed to be 5.8 X lo-l2 M at
a-GLYCEROPHOSPHATE
0
DEHYDROGENASE
06
04
02
1. 5
137
PROPERTIES
I07 ml /mole
FIG. 8. Lineweaver-Burk plot of reaction velocity against DPN concentration. The reaction mixture contained 1 ml. of 1.69 X 10-Z M sodium L-cy-glycerophosphate (in 0.2 M phosphate buffer at pH 7.00), 1 ml. of DPN solution ranging from 1.9 X 10-G M to 3.5 X 10e6 M, 0.9 ml. of distilled water, and 0.1 ml. of enzyme solution containing 54 fig. protein/ml., at 23.5”C. TABLE
III
Maximal Velocity of Reactions and Derived Michaelis Constants for Various Reactants in the Reduction of Dihydroxyacetone Phosphate by DPNH Catalyzed by Soluble a-Glycerophosphate Dehydrogenase Reactant
a-Glycerophosphate Dihydroxyacetone Dihydroxyacetone DPN
PH
phosphate phosphate
7.00 7.00 7.42 7.00
Maximal velocity, moles~l.fsec.
5.7 4.0 1.3 5.2
x x x X
IO-6 10-4 IO-4 10-e
V,
Michaelis constant, Km M
1.1 4.6 2.5 3.8
x x X x
10-d 10-a lo+ 10-4
25°C. (Table V), which agrees well with the result obtained by Burton and Wilson (8). Their average value for the equilibrium constant at 25°C. in an ionic strength of about 0.03 was 5.5 X lo-l2 M. The equilibrium constant is inversely proportional to temperature, and the slope of the line when -R(ln K) is plotted against l/T gives the
138
YOUNG
AND
PACE
TABLE
IV
Rate of Reduction of 2.0 X iO-4 M Dihydroxyacetone Phosphate and the Turnover Number of or-Glycerophosphate Dehydrogenase at pH 7.0 and at Various Temperatures
In each experiment, 1.6 pg. of enzyme was used. Turnover No. Temperature A log (lo/l) in first half min. ?noles/min. jmole enrrymc
“C.
0.041 0.302 1.017
3.0 23.1 42.0
Equilibrium
Constant
TABLE V for the Oxidation of A-wGlycerophosphate
Initial IQglycerophosphate concentration (X 10” M)
Initial DPN concentration (X 10-r M)
Initial DPNH concentration (X 10’ do
pH of 0.2 Y phT&a:e
0.0204 0.0170 0.0155 0.0301
2.44 2.44 2.44 2.44
3.37 3.37 3.37 3.37
3,650 27,000 90,700
at R4.6”C. Equilibrium constant (X lo-‘pY)
5.76 5.02 5.91 6.49
6.95 6.85 6.70 7.24
Mean 5.79 53-
I
I
I
0 Present doto a Datum from Boronowski 0 Datum from Burton and Wilson
a \
52 -
Y 5 a I 0 0
51 -
l \
50
FIG.
32
9. Relationship
I 33
I 34 l/T
I 35
\
(X 104)
between temperature and equilibrium oxidation of cu-glycerophosphate.
constant
for the
a-GLYCEROPHOSPHATE
DEHYDROGENASE
PROPERTIES
139
value of AH. In order to establish the relationship in the present instance, t,he effect of temperature on the equilibrium constant was studied at 10, 22, and 36”C., as shown in Fig. 9. For the reactions at IO, 28, and 36”C., the reaction mixtures were incubated in a water bath for 20 min. The reaction mixt’ures were then transferred immediately into the cuvette of the spectrophotometer, and the optical densit’y of each mixture was measured. The scatter of the points about the line in Fig. 9 is probably due mainly to uncontrolled slight shifts in temperature, and is large enough t,o invalidate an estimate of AH for the slope. In any case, Fig. 9 does show t,hat the equilibrium constant decreases with temperature, and at 37°C. it is approximately two-thirds of its value at room temperature. I’herefore, under equilibrium conditions the formation of ac-glycerophosphate isfavored at body temperature to an even larger degree t’han under laboratory room temperature conditions. The free energy change involved in the reduction of dihydroxyacetone phosphate to a-glycerophosphate could be calculated from the cquilibrium constant t’o be -15,300 cal./mole at 25°C. Discussion As described above, the crystalline enzyme prepared in this laboratory was apparently slightly different from that prepared by Baranowski (4), as judged by crystal shape and the ultraviolet extinction coefficients. The difference in crystal shapes could be due to slightly different conditions of crystallization, even though both were prepared by the same procedure. The enzyme crystals prepared in this study were uniform, and the ultracentrifuge patterns consistently exhibited sharp peaks which were symmetrical. The extinction coefficients at all wavelengths were somewhat lower than those reported by Baranowski (4) and by Beisenherz et al. (5). This may have been due to the fact that different methods for determining enzyme protein concentration were used by the different laboratories. Baranowski used the light absorption at 280 rnp to determine the prot’ein concentration, Beisenherz et al. used a total nitrogen determination, and in this study the biuret method, with bovine albumin as the standard, was employed. The question of the purity of any protein preparation is classically a difficult one to answer. In the present instance, while there is no direct assurance that only one molecular species comprised the preparation, at least two of the criteria for purity were met satisfactorily: The enzyme
140
YOUNG AND PACE
was obtained as uniform crystals and the ultracentrifuge sedimentation patterns were sharp. It was assumed, therefore, that the crystalline enzyme used in the present experiments was not seriously contaminated with impurities. Even if slight contamination by other proteins had been present, the physical properties examined probably would not have been affected to any great extent. Thus, as a first approximation, the values for the physical characteristics of ar-glycerophosphate dehydrogenase presented here are in all probability valid ones. The equilibrium constant for the oxidation of Lu-glycerophosphate catalyzed by soluble or-glycerophosphate dehydrogenase indicates that the probability of the occurrence of this reaction is very slight. It should be noted, however, that in addition to the presence of the soluble enzyme there also exists the particulate cr-glycerophosphate dehydrogenase, which catalyzes the oxidation of a-glycerophosphate to dihydroxyacetone phosphate in a different reaction involving no DPN. Green (2) showed that the interconversion of a-glycerophosphate and dihydroxyacetone phosphate catalyzed by the particulate a-glycerophosphate dehydrogenase at pH 7.2 greatly favors the formation of dihydroxyacetone phosphate, in contrast to the reaction catalyzed by the soluble enzyme and involving DPN. Figures 4 and 5 show clearly, with the soluble a-glycerophosphate dehydrogenase at pH 7.0, that the rate of reduction of dihydroxyacetone phosphate was about 100 times faster than the rate of oxidation of a-glycerophosphate when the same concentration of substrates was used. Therefore, the soluble enzyme appears to favor the reduction process. On the other hand, when a comparison is made of the rate of oxidation of a-glycerophosphate catalyzed by the soluble enzyme and the particulate enzyme, the rate of oxidation catalyzed by the latter is 200 times faster. Thus the particulate enzyme would appear to favor the oxidation process. From the considerations discussed above it may be postulated that in the living cell the particulate or-glycerophosphate dehydrogenase catalyzes mainly the oxidation of ar-glycerophosphate, while the soluble a-glycerophosphate dehydrogenase catalyzes mainly the reduction of dihydroxyacetone phosphate. Studies in more complex systems are necessary, however, in order to clarify this point. SUMMARY
a-Glycerophosphate dehydrogenase was crystallized as rectangular plates, having the following properties: SZO, W = 6.5 X lo-l3 sec., P =
a-GLYCEROPHOSPHATE
DEHYDROGENASE
PROPERTIES
141
0.75 ct./g. and f/j,, = 1.4. The molecular weight has been estimated to be 173,000 g./mole. a-Glycerophosphate dehydrogenase was found to be relatively thermolabile, and is most stable at pH 5.7. Approximate Michaelis constants at pH 7.0 were found as follows: for cY-glycerophosphate, 1.1 X lop4 M; for dihydroxyacetone phosphate, 4.6 X lo4 M; for DPN, 3.8 X 10W4M. The equilibrium constant for the oxidation of cr-glycerophosphate was found to be 5.8 X lo-l2 M at 25°C. All the properties studied indicate that within the pH range 7-8 the reaction favors formation of a-glycerophosphat,e. REFERENCES
1. 2. 3. 4. 5.
MEYERHOF, O., Arch. ges. Physiol., PjZtiger’s 176, 20 (1919). GREEN, D. E., Biochem. J. 80, 629 (1936). EULER, H. v., ADLER, E., AND GUENTHER, G., 2. physiol. Chem. 249, 1 (1937). BARANO~SKI, T., J. Biol. Chem. 180, 535 (1949). BEISENHERZ, G., BOLTZE, I-1. J., BUTCHER, T. H., CZOK, R., GARBADE, K. M., MEYER-ARENDT, E., AND PFLEIDERER, G., Z. Naturforsch. 8b, 555 (1953). 6. EULER, H. v., ADLER, E., GUNTHER, G., AND HELLSTROM, H., Z. physiol. Chem. 246, 217 (1937). 7. GREEX, D. E., NEEDHAM, D. M., AND DEWAN, J. G., Biochem. J. 31, 2327 (1937).
8. BURTON, K., AND WILSON, T. H., Biochem. J. 64, 86 (1953). 9. KIXG, H., AND PYMAN, F. L., J. Chem. Sot. 106, 1238 (1914). 10. MEYERHOF, O., AND LOHMANN, K., Biochem. Z. 271, 89 (1934). Il. STUMPF, I-‘. K., J. Biol. Chem. 176, 233 (1948). 12. KIRK, P. I,., “Quantitative Ultramicroanalysis,” p. 287. John Wiley & Sons, Inc., New York, 1950. 13. BARANOWSKI, T., AND NIEDERLAND, T. R., J. Biol. Chem. 180, 543 (1949). 14. DAVIES, D. R., Biochem. J. 28, 529 (1934). 15. BEISENHERZ, G., B~CHER, T., AND GARBADE, K., Methods in Enzymol. 1, 391 (1955).
16. (a) SVEDBERG, T., AND PEDERSEN, K. O., “The Ultracentrifuge,” p. 273. Oxford University Press, London, 1940; (b) ibid., p. 57. 17. ALEXANDER, A. E., AND JOHNSON, P., “Colloid Science,” p. 351. Oxford University Press, London, 1949. 18. ONCLEY, J. I,., Ann. N. Y. Acad. Sci. 41, 121 (1941). 19. MICHAELIS, L., Biochem. 2. 234, 139 (1931). 20. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66, 658 (1934). 21. ALBERTY, R. A., Advances in Enzymol. 17, 1 (1956).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
76,
125-141 (1958)
Some Physical and Chemical Properties of Crystalline ~Glycerophosphate Dehydrogenase’ Ho Lee Young and Nello Pace From the Department
of Physiology, School of Medicine, Rerkeley, California Received
September
Cniversit!l
of California,
19, 1957
INTRODUCTION
a-Glycerophosphate dehydrogenase act,ivity was first observed by Meyerhof (1) in 1919, and since that time two distinct a-glycerophosphate dehydrogenases have been isolated from mammalian muscle. The first, which is apparently associated with the cell particulates and which does not require the presence of diphosphopyridine nucleotide (DPN) for its action, was isolated and studied thoroughly by Green (2) an 1936. Von Euler and co-workers (3) in 1937 isolated from rabbit muscle another cY-glycerophosphate dehydrogensse which does require DPN as a hydrogen acceptor, and which has been termed the “soluble” Lu-glycerophosphate dehydrogenase, t’o distinguish it from the “particulate” a-glycerophosphate dehydrogenase of Green. It is t’he soluble cr-glycerophosphate dehydrogenase with which t,he present study is concerned. The soluble enzyme has been crystallized previously by Baranowski (4) and Beisenherz et al. (5); however, information on the physical properties of crystalline soluble a-glycerophosphate dehydrogenase is sparse, and the present paper describes several additional physical properties of the enzyme. Chemical properties of soluble a-glycerophosphate dehydrogenase have also been studied previously by several workers. The equilibrium constant for the oxidation of a-glycerophosphate has been measured by von Euler et al. (3, 6), Green et al. (7), Baranowski (4), and Burton and Wilson (8). The specificity of the enzyme and the Michaelis constant for t,he oxidation of Lu-glycerophosphate t’o dihydroxyacetone phosphat,e 1 These studies were supported 222(31) between the Office of Naval
under Contracts K7onr-295(04) and NonrResearch and t,he University of California. 125
126
YOUNG
AND
PACE
has been determined by Green et al. (7). The present study represents an examination of these and other chemical properties of the crystalline enzyme. MATERIAL
AND
METHODS
Reagents 1. D, L-or-Glycerophosphate and L-a-Glycerophosphate. Sodium D,L-a-glycerophosphate was made according to the method described by King and Pyman (9). The barium L-u-glycerophosphate used was kindly donated by Dr. E. Baer of the University of Toronto. The latter salt was dissolved directly in distilled water for low concentrations, but for high concentrations it was converted to the potassium salt immediately before it was used in an experiment. 2. Dihydroxyacetone Phosphate. The calcium salt was prepared enzymically by the method of Meyerhof and Lohmann (10) and was stored under vacuum in the cold for not longer than 2 weeks. The amount of dihydroxyacetone phosphate was determined immediately before use by the iodine oxidation and alkali-labile phosphate determination as described by Stumpf (11). The freshly prepared dihydroxyacetone phosphate contained an average of 90% dihydroxyacetone phosphate, a maximum of 0.5yo inorganic phosphate, and approximately 10% other phosphate, which probably consisted mostly of hexose diphosphate and glyceraldehyde phosphate. None of these impurities interferes with the assay of the crystalline enzyme. When low concentrations of dihydroxyacetone phosphate were required, the calcium salt was used directly; however, when high concentrations were needed, it was converted to the potassium salt. 3. Other Chemicals. Double glass-distilled water was used. Ca hexose diphosphate (Schwarz Laboratories, Inc.), DPN, and DPNH (Sigma Chemical Co.) and bovine albumin (Armour Laboratories) were also used.
Enzyme Assay Methods 1. Assay Method I (Oxidation of DPNH). This method is based on the oxidation of DPNH by dihydroxyacetone phosphate in the presence of cu-glycerophosphate dehydrogenase to form a-glycerophosphate and DPN, as described by Baranowski (4). The amount of DPNH oxidized and the rate of the oxidation were determined by measuring the change in light absorption at 340 rnr, using a Beckman model DU spectrophotometer. Readings of the spectrophotometer were made every 30 sec. until the reaction was essentially complete, usually u-ithin 5 min. after the start of the reaction. 2. Assay Method II (Reduction of DPN). This assay was based on the reverse of the chemical reaction described in the preceding section. The rate of appearance of DPNH in the reaction mixture was measured by following the change in light absorption at 340 mp.
Protein Determination The concentration biuret method (12).
of protein
in the enzyme solutions
was determined
by Kirk’s
a-GLYCE~OPHOSPH.4TE DEHYDHOGES.4SE PIZOPEHTIES
I’Ic:. I. wGlyc:erophosphate
dehydrogenase
crystals
from
rabbit
muscle
127
(2&5X).
RESULTS
C‘r+stallixation
of cY-Glycerophosphate Dehydrogenase
The prcparut~ion of myogen X crystals from rabbit muscle was carried out by the met’hod described by Baranowski and Kiederland (13) wit,h slight modification, to separate the sediment,ed protein by wntrifugntion inst,ead of by filtration. Preparation of crystalline a-glycerophosphate dehydrogenase was performed as described by Baranowski (4) ; however, the crystals obtained in this study were rectangular plates, as shown in Fig. 1, lvhilr those prepared by Buranowski were rhombic plates. The crystalline errzyme was stable for several weeks when it’ was stored as a suspension in npproximately 50 7%(KH,)$30., in the cold. Aldolase activity could trot’ be det ected by thr method of Baranowski and Sicderland (I:<). Phosphatasr acti\4ty lilwwisc was not detectable by the technique of Da\-ier (II).
The ultjraviolct absorption spectrum from 210 to 300 mp ~w:: generally mow similar to that measured by Baranowski (4) thalr to that of Beisrw hcrz cl rtl. (15), but exhibited some differences front Iwth. ‘l’hc present
128
YOUNG
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PACE
preparation had a somewhat sharper absorption peak around 280 rnp, and, in general, the extinction coefficients at all wavelengths were from 2 to 20% lower than those reported by Baranowski. Beisenherz et al. (15) found that the maximal absorption of the enzyme was at 270 rnp, whereas the present preparation showed a maximal absorption at 278 rnp. At 270 rnp the extinction coefficient of t,he present preparation was about 20% lower than that of the Beisenherz preparation. Sedimentation Coej’kient a-Glycerophosphate dehydrogenase crystals were filtered from their (NHh)$304 suspension by suction through Whatman No. 50 filter paper. Samples of the relatively dry crystals were dissolved in a small amount of water and dialyzed against 0.5 M (NHJzS042 for 18 hr. in the cold. The cr-glycerophosphate dehydrogenase solutions were centrifuged in a Spinco model E ultracenbrifuge analyt’ical rotor at 59,780 r.p.m., an average centrifugal field of 260,000 X g, with refrigeration. The sedimentation coefficient, under the standard condition of pure water at 2O”C., szo, -a., was calculated by the Svedberg and Katsurai (16a) equations. An ultracentrifuge pattern of a-glycerophosphate dehydrogenase is shown in Fig. 2. The sedimentation coefficients calculated from the upper and lower patterns are 6.4 X IO-l3 sec. and 6.5 X lo-l3 sec., respectively. Sedimentation coefficients were det’ermined for several protein concentrations of two separate enzyme preparations, and the values of ~20,w were plotted against the protein concentrations for each preparation. The szn, ,” value was only little affected by change in protein concentration in the range studied from 0.2 mg./ml. to 10 mg./ml. The zeroprotein-concentration extrapolated values of szo, w were found to be 6.4 X lo-l3 sec. and 6.6 X lo-l3 sec., respectively. The difference was regarded as not significant within the limits of accuracy of the method; hence the mean value of 6.5 X lo-l3 sec. was taken to represent the sedimentation coefficient of a-glycerophosphate dehydrogennse in water at 2O’C. * Ammonium sulfate, 0.5 84, instead of KC1 was used to prevent the electrostatic effect on particle sedimentation. The wglycerophosphate dehydrogenawe precipitated out and did not redissolve in water when it was suspended in 0.5 M KCl. Two possible explanations for this behavior may be mentioned: either the ionic strengt,h of 0.5 M KC1 may have been too low, or the enzyme was denatured by either or both the potassium and chloride ions.
FIG. 2. Ultracentrifuge patterns of ru-glycerophosphate dehydrogenase as a function of time. The dialyzed cnzymc solution in the upper patt,ern contained 2.7 mg. protein/ml. of 0.5 M (NHI)&304 , and the dialyzed solution in the lower pattern contained 0.51 mg. protein/ml. of 0.5 M (NHa)280a , both at, pH 5.6. The rotor speed was 5!),780 r.p.m. and the Itar angle was 60”. The times of each frame were 0, 8, 16, 24, and 32 min. after rcachillg full sl~cctl. ‘I’hc initial 1enipcraturr ~vas 6.0”( ‘_ and t hcl final tcmpcraturc kvas 8.4”(‘.
s
3 ? Fs
130
YOUNG
AND
spparent
PACE
SpeciJic Volume
The apparent specific volume, 7, determination was carried out by weighing the solvent, 1 M (NHI) $04 , alone and then weighing an a-glycerophosphate dehydrogenase solution of 0.0040 g./cc. in a pycnometer containing 0.9906 ml. at 24°C. The enzyme solution was previously dialyzed against 1 M (NHI)$04 . The apparent specific volume was calculated by means of the equation derived by Kraemer (16b). The weight of the dialyzed 0.0040 g./cc. enzyme solution, m, was 0.1063 g. The weight fraction of the dialyzed enzyme solution (~1 = gv/m) was 0.0037, the weight fraction of the solvent (WZ = 1 - ~1) was 0.9963, and the specific volume of solvent (V,) was 0.9328 ct./g. The results are shown in Table I, and the mean apparent specific volume of a-glycerophosphate dehydrogenase from five determinations was found to be 0.75 ct./g. TABLE
I
Mean of Five Deternlinations
of the Apparent Specific Volamz of a-Glycerophosphate Dehydrogenase A pycnometer containing 0.09906 ml. (v) at 24°C. was used.
Determination
1 2 3 4 5
Weight
of 1 M (NH&SOa
(mo)
Specific solution
0.1060 0.1060 0.1060 0.1061 0.1060
volume
of
(V = v/m)
Apparent specific volume of a-glycerophosp&te dehydrogenase (V)
0.9319 0.9319 0.9328 0.9319 0.9328 Standard
Mean deviation f
0.70 0.70 0.95 0.70 0.70 0.75 0.11
viscosity The enzyme solutions used for viscosity measurements were prepared in the same way as the one used for the apparent specific volume study. The flow time for the solvent and the enzyme solutions was determined five times in a microviscometer of the Ostwald type and expressed as an average for each solution, as shown in Table II. The relative viscosity, qr , and the ratio of specific viscosity, vsD, to the volume fraction of the solute were calculated by the methods described by Alexander and Johnson (17). The results are shown in Table II. Mean values for as,/+ of 8.6 and 10.0 were found for the two concentrations of a-glycerophosphate dehydrogenase. Because of the limited amount of crystalline en-
a-CLYCEROPHOSPHATE
DEHYDROGENASE
131
PROPERTIES
Mean oj” Five Determinations of the IZatio of SpeciJic Viscositll to Volume Fraction (Q,/+) at ~5.5%‘. 0.407,
Determindtion
solution
Flow time of 1 ‘K (hTHh)nS0~ !I?)
Flow time of en7.yme solution (11)
0.807~ solution
dchydrogenase
of a-glycero hosphate in 1 M ( ff H&SOa
dehydrogenase
Relative viscosity enzyme solution (WY
of
Ratio of specific viscosity to volume fraction (?.P/Sbi”
Flow time of enzyme solution ill)
SK. 1
2 3 4 5 Mean s. D.
of a-glycerophosphate in1 .44 (KH~)zSOI Relative viscosity of enzyme solution (4ri
Ratio of specific viscosity to volume fraction f?SP/V+)
1.060 1.060 1.060 1.060 1.059 1.060 fO.0005
10.0 10.0 10.0 10.0 0.8 10.0 fO.10
SM.
71.43 il.35 71.33 il.45 71.42 71.40 f0.053
73.18 73.16 73.18 73.16 73.1-I 73.16 f0.014
1.026 1.026 1.026 1.026 1.025 1.026 fO.0005
I’ 7r = pltl/pnt2 , where deusity of 1 112 (KH,)&Wd h ll.Y,/@ = or -
8.7 X.7 8.7 8.7 8.3 8.6 f0.17
of enzyme
solution,
75.58 75.59 75.58 i5.48 75.57 75.56 fO.045
p, . was l.Oi3,
and density
, p2 , \vas 1.072. 1.
4
zyme available, only these two concentrations were studied. However, the value of qlsP/+ at infinite dilution was obtained by extrapolation to $ = 0 from the two values obtained, when qs,,/$ was plotted against 4, and was found to be 7.2. This is probably a reasonable approximation of the true ratio. From the viscosity data a frictional ratio, f/j” , of 1.4 was obtained from the Oncley contour diagrams (18), using the value of qsP/4 of 7.2. The molecular weight, M, could then be computed by the following equnt8ion : M2’3 = (f/f,,) (Gav,sN),‘(l
- VP,) (3v/47rN)1’3
where qWis the viscosity of water; N is the Avogadro number, 6.0235 X 10z3/mole; and pa is the density of 0.5 M (NHd)zSO., . The molecular weight by this calculation was found t,o be 173,000 g.,/mole. Stability
of the Enzyme
Both thermal stability and the pH stability were examined. The enzyme was found to be relatively thermolabile, 53 % of it,s activity being lost by heating to 55°C. for 1 min., and nearly all activity being lost at
132
YOUNG
AND
PACE
60°C. However, there was no appreciable loss of activity when it was kept at room temperature, 22”C., for as long as 30 min. The pH stability was studied at two different temperatures, 1.5 and 28”C., using barbital buffer solutions (19). All the solutions were kept at the particular pH and temperature for 2 hr. before testing their activity. The results are shown in Fig. 3, and the highest rate at pH 6 was taken as 100 %. The enzyme was most stable around pH 5.7, both in the cold and at 28°C. The protein was precipitated by extreme acid or alkali concentrations at both temperatures, and the precipitated protein was no longer soluble in water. Another experiment was done to test the stability of the enzyme. A solution containing 1 mg. protein/ml. in approximately 0.2 N ammonium sulfate at pH 5.8 was kept between 0 and 4°C. The activity of the enzyme was measured at intervals during a 2-week period. It was found that there was no change in enzyme activity during this time. Curves
pH-Activity
The effect of pH on the reduction of dihydroxyacetone phosphate is shown in Fig. 4, and the highest rate of the reaction was found at ap-
----k-g
4
PH
6
6
FIG. 3. Effect of pH on the stability of two portions of an a-glycerophosphate dehydrogenaae solution: one kept at 1.5”C. and the other kept at 28°C.
WGLYCEROPHOSPHATE
j.; -
65
DEHYDROGENASE
PROPERTIES
,
,
;
70
75
80
133
8
PH
FIG. 4. The effect of pH on the rate of reduction of dihydroxyacetone phosphate in the presence of a-glycerophosphate dehydrogenase. Dihydroxyacetone phosphate, 2.7 X lo-” M, WE used as the substrate.
pH 7.5. The effect of pH on the oxidation of L( -))cr-glycerois shown in Fig. 5, and the highest rate of oxidation occurred at about pH 10.2. For these curves 0.2 M phosphate buffer was used to obt,ain the pH values between 6.7 and 8.6, and 0.03 M glycine buffer was used in the pH range from 1.5 to 3.5 and from 8.5 to 11.5. proximately phosphate
Michaelis Constant In the present instance, where five components are involved, application of the Lineweaver and Burk treatment (20) to the kinetic data obtained by varying the concentration of one component at a time leads only to approximate values for V, and K, for each component, and hence complete determination of the constants cannot be made (21). The values obtained, however, are useful in establishing gross characteristics of the reaction involved. The relationship between the rate of reaction and the concentration of different substrates is shown in Figs. 6 and 7, while the relationship between the rate of reaction and DPN concentraCon is shown in Fig. 8.
134
YOUNG
AND
PACE
FIG. 5. The effect of pH on the rate of oxidation of L-ol-glycerophosphate in the presence of a-glycerophosphate dehydrogenase. a-Glycerophosphate dehydrogenase, 3.5 X 10-S M, was used as the substrate.
The maximal velocity for each reaction, obtained from Figs. 6-8, and the calculated Michaelis constants are given in Table III. Table III shows that the Michaelis constant, K, , for the oxidation of a-glycerophosphate is 1.1 X 10e4 M. This value is low in comparison with the one reported by Green (7) of 1.25 X 10e3 M. In the Lineweaver-Burk plot of reaction velocity against DPNH concentration, all points were fairly scattered. A reliable estimated Michaelis constant for DPNH could not be obtained; however, its concentration effect appeared to be much less. Table IV shows the marked dependence on temperature of the rate of reduction of dihydroxyacetone phosphate, and hence of the value for the turnover number for cu-glycerophosphate dehydrogenase. Thus in any comparison of values for turnover number obtained by different investigators, the substrate concentration, the pH, and the temperature must be taken into account. Baranowski (4) has reported a turnover number of 45,800 moles sub-
a-GLYCEX~OPHOSPHA!l’E
DEHYDROGENASE
I T
10’
PROPERTIES
135
ml. /mole
FIG. 6. Lineweaver-Rurk plot of reaction velocity against r,-a-glgcerophosphate concentratjion at, pH 7.00. The reaction mixture, totaling 3 ml., contained 1 ml. of 2.44 X 10F3 .?I Dl”?;T (in 0.2 M phosphate buffer at pH 7.00), I ml. of sodium or-glycerophosphste solution ranging from 1.2 X 10-d M to 5.7 X UP M, 0.9 ml. of distilled water, and 0.1 ml. of enzyme solution containing 140 pg. protein/ml. The temperature at which the reactions were measured was 23.3”C.
strate/miu./lOO,OOO g. protein for a-glycerophosphatje dehydrogenase, using 0.28 M dihydroxyacetone phosphat’e as t’he sub&ate at pH 7.0 nlld 22”C., and Beisenherz et al. (15) reported an estimated turnovel number of 51,900 at 25°C. From t,he Michaelis constant of 4.6 X 10m4&l for the reduction of dihydroxyacetone phosphate shown in Table III, a turnover number of 84,400 moles/min./mole of enzyme at pH 7.0 and 22°C. may be computed. The DPNH used in determining K, for dihydroxyaretone phosphate was about double the amount used by Baranomski for turnover study. Thus t,he turnover number for the crystalline enzyme preparutSion used in the present study at, the same pH would appear to he substant,ially greater than that obtained by either Barnnowski or Beisenherz et al. ; however, such a comparison may not be valid because of the widely differing sub&rate concentrabions used in the two studies.
136
YOUNG
AND
PACE
62 E . ri :: 2 E
$ -I>
540
/
32,000
;Pp 0
I
2
I 5
IO’
I 4 ml./mols
I 6
a
O/ a/ 0
6-
I
0 0
0.5
( r
I I.0
I 1.5
, 2.0
IO' ml. /mole
7. Lineweaver-Burk plot of reaction velocity against dihydroxyacetone phosphate concentration. (a) Upper graph: The reaction mixture contained 1 ml. of 6.15 X 1O-4 M DPNH (in 0.2 M phosphate buffer at pH 7.00), 1 ml. of sodium dihydroxyacetone phosphate solution ranging from 1.0 X 10e6 M to 3.0 X lO+ M, 0.9 ml. of distilled wat.er, and 0.1 ml. of enzyme solution containing 200 pg. protein/ml., at 23°C. (b) Lower graph: The reaction mixture contained 1 ml. of 7.35 X lo-” M DPNH (in 0.2 M phosphate buffer at pH 7.42), 1. ml. of dihydroxyacetone phosphate solution, ranging from 1 .O X 10e6 M to 3.0 X 10e3 M, 0.9 ml. of distilled water, and 0.1 ml. of enzyme solution containing 20 pg./ml., at 24°C. FIG.
Equilibrium
Constant
The equilibrium constant for the reduction of dihydroxyacetone phosphate has been measured by von Euler (3, 6), Green (7), Baranowski (4), and Burton and Wilson (8). However, their results are not consistent. In the present work the equilibrium constant was measured by assay method II at 24.5%. and was computed to be 5.8 X lo-l2 M at
a-GLYCEROPHOSPHATE
0
DEHYDROGENASE
06
04
02
1. 5
137
PROPERTIES
I07 ml /mole
FIG. 8. Lineweaver-Burk plot of reaction velocity against DPN concentration. The reaction mixture contained 1 ml. of 1.69 X 10-Z M sodium L-cy-glycerophosphate (in 0.2 M phosphate buffer at pH 7.00), 1 ml. of DPN solution ranging from 1.9 X 10-G M to 3.5 X 10e6 M, 0.9 ml. of distilled water, and 0.1 ml. of enzyme solution containing 54 fig. protein/ml., at 23.5”C. TABLE
III
Maximal Velocity of Reactions and Derived Michaelis Constants for Various Reactants in the Reduction of Dihydroxyacetone Phosphate by DPNH Catalyzed by Soluble a-Glycerophosphate Dehydrogenase Reactant
a-Glycerophosphate Dihydroxyacetone Dihydroxyacetone DPN
PH
phosphate phosphate
7.00 7.00 7.42 7.00
Maximal velocity, moles~l.fsec.
5.7 4.0 1.3 5.2
x x x X
IO-6 10-4 IO-4 10-e
V,
Michaelis constant, Km M
1.1 4.6 2.5 3.8
x x X x
10-d 10-a lo+ 10-4
25°C. (Table V), which agrees well with the result obtained by Burton and Wilson (8). Their average value for the equilibrium constant at 25°C. in an ionic strength of about 0.03 was 5.5 X lo-l2 M. The equilibrium constant is inversely proportional to temperature, and the slope of the line when -R(ln K) is plotted against l/T gives the
138
YOUNG
AND
PACE
TABLE
IV
Rate of Reduction of 2.0 X iO-4 M Dihydroxyacetone Phosphate and the Turnover Number of or-Glycerophosphate Dehydrogenase at pH 7.0 and at Various Temperatures
In each experiment, 1.6 pg. of enzyme was used. Turnover No. Temperature A log (lo/l) in first half min. ?noles/min. jmole enrrymc
“C.
0.041 0.302 1.017
3.0 23.1 42.0
Equilibrium
Constant
TABLE V for the Oxidation of A-wGlycerophosphate
Initial IQglycerophosphate concentration (X 10” M)
Initial DPN concentration (X 10-r M)
Initial DPNH concentration (X 10’ do
pH of 0.2 Y phT&a:e
0.0204 0.0170 0.0155 0.0301
2.44 2.44 2.44 2.44
3.37 3.37 3.37 3.37
3,650 27,000 90,700
at R4.6”C. Equilibrium constant (X lo-‘pY)
5.76 5.02 5.91 6.49
6.95 6.85 6.70 7.24
Mean 5.79 53-
I
I
I
0 Present doto a Datum from Boronowski 0 Datum from Burton and Wilson
a \
52 -
Y 5 a I 0 0
51 -
l \
50
FIG.
32
9. Relationship
I 33
I 34 l/T
I 35
\
(X 104)
between temperature and equilibrium oxidation of cu-glycerophosphate.
constant
for the
a-GLYCEROPHOSPHATE
DEHYDROGENASE
PROPERTIES
139
value of AH. In order to establish the relationship in the present instance, t,he effect of temperature on the equilibrium constant was studied at 10, 22, and 36”C., as shown in Fig. 9. For the reactions at IO, 28, and 36”C., the reaction mixtures were incubated in a water bath for 20 min. The reaction mixt’ures were then transferred immediately into the cuvette of the spectrophotometer, and the optical densit’y of each mixture was measured. The scatter of the points about the line in Fig. 9 is probably due mainly to uncontrolled slight shifts in temperature, and is large enough t,o invalidate an estimate of AH for the slope. In any case, Fig. 9 does show t,hat the equilibrium constant decreases with temperature, and at 37°C. it is approximately two-thirds of its value at room temperature. I’herefore, under equilibrium conditions the formation of ac-glycerophosphate isfavored at body temperature to an even larger degree t’han under laboratory room temperature conditions. The free energy change involved in the reduction of dihydroxyacetone phosphate to a-glycerophosphate could be calculated from the cquilibrium constant t’o be -15,300 cal./mole at 25°C. Discussion As described above, the crystalline enzyme prepared in this laboratory was apparently slightly different from that prepared by Baranowski (4), as judged by crystal shape and the ultraviolet extinction coefficients. The difference in crystal shapes could be due to slightly different conditions of crystallization, even though both were prepared by the same procedure. The enzyme crystals prepared in this study were uniform, and the ultracentrifuge patterns consistently exhibited sharp peaks which were symmetrical. The extinction coefficients at all wavelengths were somewhat lower than those reported by Baranowski (4) and by Beisenherz et al. (5). This may have been due to the fact that different methods for determining enzyme protein concentration were used by the different laboratories. Baranowski used the light absorption at 280 rnp to determine the prot’ein concentration, Beisenherz et al. used a total nitrogen determination, and in this study the biuret method, with bovine albumin as the standard, was employed. The question of the purity of any protein preparation is classically a difficult one to answer. In the present instance, while there is no direct assurance that only one molecular species comprised the preparation, at least two of the criteria for purity were met satisfactorily: The enzyme
140
YOUNG AND PACE
was obtained as uniform crystals and the ultracentrifuge sedimentation patterns were sharp. It was assumed, therefore, that the crystalline enzyme used in the present experiments was not seriously contaminated with impurities. Even if slight contamination by other proteins had been present, the physical properties examined probably would not have been affected to any great extent. Thus, as a first approximation, the values for the physical characteristics of ar-glycerophosphate dehydrogenase presented here are in all probability valid ones. The equilibrium constant for the oxidation of Lu-glycerophosphate catalyzed by soluble or-glycerophosphate dehydrogenase indicates that the probability of the occurrence of this reaction is very slight. It should be noted, however, that in addition to the presence of the soluble enzyme there also exists the particulate cr-glycerophosphate dehydrogenase, which catalyzes the oxidation of a-glycerophosphate to dihydroxyacetone phosphate in a different reaction involving no DPN. Green (2) showed that the interconversion of a-glycerophosphate and dihydroxyacetone phosphate catalyzed by the particulate a-glycerophosphate dehydrogenase at pH 7.2 greatly favors the formation of dihydroxyacetone phosphate, in contrast to the reaction catalyzed by the soluble enzyme and involving DPN. Figures 4 and 5 show clearly, with the soluble a-glycerophosphate dehydrogenase at pH 7.0, that the rate of reduction of dihydroxyacetone phosphate was about 100 times faster than the rate of oxidation of a-glycerophosphate when the same concentration of substrates was used. Therefore, the soluble enzyme appears to favor the reduction process. On the other hand, when a comparison is made of the rate of oxidation of a-glycerophosphate catalyzed by the soluble enzyme and the particulate enzyme, the rate of oxidation catalyzed by the latter is 200 times faster. Thus the particulate enzyme would appear to favor the oxidation process. From the considerations discussed above it may be postulated that in the living cell the particulate or-glycerophosphate dehydrogenase catalyzes mainly the oxidation of ar-glycerophosphate, while the soluble a-glycerophosphate dehydrogenase catalyzes mainly the reduction of dihydroxyacetone phosphate. Studies in more complex systems are necessary, however, in order to clarify this point. SUMMARY
a-Glycerophosphate dehydrogenase was crystallized as rectangular plates, having the following properties: SZO, W = 6.5 X lo-l3 sec., P =
a-GLYCEROPHOSPHATE
DEHYDROGENASE
PROPERTIES
141
0.75 ct./g. and f/j,, = 1.4. The molecular weight has been estimated to be 173,000 g./mole. a-Glycerophosphate dehydrogenase was found to be relatively thermolabile, and is most stable at pH 5.7. Approximate Michaelis constants at pH 7.0 were found as follows: for cY-glycerophosphate, 1.1 X lop4 M; for dihydroxyacetone phosphate, 4.6 X lo4 M; for DPN, 3.8 X 10W4M. The equilibrium constant for the oxidation of cr-glycerophosphate was found to be 5.8 X lo-l2 M at 25°C. All the properties studied indicate that within the pH range 7-8 the reaction favors formation of a-glycerophosphat,e. REFERENCES
1. 2. 3. 4. 5.
MEYERHOF, O., Arch. ges. Physiol., PjZtiger’s 176, 20 (1919). GREEN, D. E., Biochem. J. 80, 629 (1936). EULER, H. v., ADLER, E., AND GUENTHER, G., 2. physiol. Chem. 249, 1 (1937). BARANO~SKI, T., J. Biol. Chem. 180, 535 (1949). BEISENHERZ, G., BOLTZE, I-1. J., BUTCHER, T. H., CZOK, R., GARBADE, K. M., MEYER-ARENDT, E., AND PFLEIDERER, G., Z. Naturforsch. 8b, 555 (1953). 6. EULER, H. v., ADLER, E., GUNTHER, G., AND HELLSTROM, H., Z. physiol. Chem. 246, 217 (1937). 7. GREEX, D. E., NEEDHAM, D. M., AND DEWAN, J. G., Biochem. J. 31, 2327 (1937).
8. BURTON, K., AND WILSON, T. H., Biochem. J. 64, 86 (1953). 9. KIXG, H., AND PYMAN, F. L., J. Chem. Sot. 106, 1238 (1914). 10. MEYERHOF, O., AND LOHMANN, K., Biochem. Z. 271, 89 (1934). Il. STUMPF, I-‘. K., J. Biol. Chem. 176, 233 (1948). 12. KIRK, P. I,., “Quantitative Ultramicroanalysis,” p. 287. John Wiley & Sons, Inc., New York, 1950. 13. BARANOWSKI, T., AND NIEDERLAND, T. R., J. Biol. Chem. 180, 543 (1949). 14. DAVIES, D. R., Biochem. J. 28, 529 (1934). 15. BEISENHERZ, G., B~CHER, T., AND GARBADE, K., Methods in Enzymol. 1, 391 (1955).
16. (a) SVEDBERG, T., AND PEDERSEN, K. O., “The Ultracentrifuge,” p. 273. Oxford University Press, London, 1940; (b) ibid., p. 57. 17. ALEXANDER, A. E., AND JOHNSON, P., “Colloid Science,” p. 351. Oxford University Press, London, 1949. 18. ONCLEY, J. I,., Ann. N. Y. Acad. Sci. 41, 121 (1941). 19. MICHAELIS, L., Biochem. 2. 234, 139 (1931). 20. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66, 658 (1934). 21. ALBERTY, R. A., Advances in Enzymol. 17, 1 (1956).