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Yeast glyoxalase I. Circular dichroic spectra and pH effects
0020-711X/86 $3.00 + 0.00 Copyright © 1986 Pergamon Press Ltd
Int. J. Biochem. Vol. 18, No. 6, pp. 549 555, 1986
Printed in Great Britain. All rights reserved
CIRCULAR
Y E A S T G L Y O X A L A S E I. D I C H R O I C S P E C T R A A N D pH EFFECTS
KENNETH T. DOUGLASI*, ANDREW P. SEDDONtt and YASHUSHI NAKAGAWA2 IDepartment of Chemistry, University of Essex, Colchester, Essex CO4 3SQ, U.K. [Tel. (0206) 862286] 2Michael Reese Hospital and Medical Center, 29th Street and Ellis Avenue, Chicago, IL 60616, U.S.A. (Received 31 October 1985) AMtraet--l. Large scale isolation and physicochemical characterisation of yeast glyoxalase I showed that this enzyme contained small amounts of carbohydrates. 2. Circular dichroic spectra of the enzyme measured in the presence and absence of S-(pbromobenzyl)glutathione indicated perturbation of a tyrosine on binding of this competitive inhibitor. 3. Values of K, for competitive inhibitors were pH invariant over the accessible pH range.
inhibition of glyoxalase I by porphyrins (Douglas et al., 1982b). This presumably implies a conformational difference in the forms of enzyme which bind substrate and inhibitor respectively. Considerable evidence of flexibility in this system has been noted (Douglas and Shinkai, 1985).
INTRODUCTION
Glyoxalase I (EC 4.4.1.5) converts the hemithiolacetal formed between glutathione (GSH) and methylglyoxal or other ~-ketoaldehydes into Slactoylglutathione etc. (Racker, 1951), which is then hydrolysed under glyoxalase II catalysis, see equation (1).
GS GSH + CH3COCHO
I
C-----O
GS
I
~-- H - - C - - - O H
I
glyoxalase I
, H---C --OH [ CH 3
C CH 3
0
GSH glyoxalas¢ , + I1
(1)
COl H-@--OH CH3
A variety of suggestions concerning the function of glyoxalase I has been made ranging from roles in cell proliferation/cancer (Szent-Gyorgi, 1968), enhancement of anti-IgE-induced histamine release (Gillespie, 1979) and microtubule assembly (Gillespie, 1975) to protection against intestinal bacteria (Aronsson et al., 1977). Our interest in this enzyme stems from many reports that glyoxalase I inhibitors often have anticancer activity (Vince and Daluge, 1971; Iio et al., 1975). The pH profile for the yeast glyoxalase I-catalysed reaction of a n u m b e r of ct-ketoaldehydes has been reported (Van der Jagt and Han, 1973). It was found that /)max was insensitive to pH, while K,~ values increased at lower and higher pH (with pK~pp values of 4.7-5.2 and 8.4-8.9, depending on the ~t-ketoaldehyde). In spite of the pH-dependence of K,, we have found that competitive inhibition constants for S-blocked and for S,N-blocked glutathiones are essentially pH-independent (Douglas et al., 1982a) in marked contrast to the pH-dependent competitive
MATERIALS AND METHODS
*Author to whom correspondence should be addressed. tPresent address: Department of Biochemistry, Cornell University Medical College, New York, NY 10021, U.S.A.
The following materials were obtained from Sigma Chemical Co. Ltd. (Dorset, England): methylglyoxal (40% aqueous solution), reduced glutathione, calibration proteins for SDS molecular weight determinations, semicarbazide hydrochloride. Tetranitromethane was from Aldrich Chemical Co. Ltd. Other commercial materials were of the highest quality available. Methylglyoxal was purified according to Han (1975) and standardized by a modification of the enzymatic method (Bergmeyer, 1974) and also by a semicarbazide procedure (Alexander and Boyer, 1971). The glyoxalase I assay system (after Racker, 1951) contained GSH (0.66mM), methylglyoxal (2mM) and KH2PO 4 (0.05 M, pH 6.60) in a final volume of 3.0 ml. This mixture was incubated at 25°C (5 min) to ensure complete equilibration to the hemimercaptal. Reaction was initiated by addition of glyoxalase I and the absorbance at 240 nm recorded against time using a Pye-Unicam SP8100 UV-visible spectrophotometer at 25°C. A unit of glyoxalase I is here defined as the amount of enzyme catalysing formation of 1 #mol of S-o-lactoylglutathione per minute under the above conditions. Protein concentrations were determined spectrophotometrically using a dye-binding procedure (Bradford, 1976; Spector, 1978) for more purified samples. Purification of Glyoxalase I from Yeast (a) Autolysis. Compressed baker's yeast (20 kg), crumbled and mixed with chloroform (41), was left with occasional
qztO
550
KENNETH T. DOUGLAS et al.
stirring for 1 hr or until the mixture liquefied. Distilled water (81) was added, the pH was adjusted to 6.6 with sodium hydroxide (I M) and readjusted after 2 hr and this suspension was left overnight at room temperature. All subsequent steps were performed at 4'C unless otherwise stated. The autolysed suspension was centrifuged (2800rpm, 40mins, MSE "Mistral 4 1" centrifuge, Head No. 43124-105). The heavy precipitate was discarded. (b) Ammonium sulphate fractionation. The supernatant was fractionated over a 40-65% (NH4)2SO 4 range. Firstly, (NH4)2SO 4 was added to the supernatant to give 40% saturation. The solution was stirred for ~ 1 hr and centrifuged (2800 rpm, 40 min). The precipitate was discarded and the supernatant brought to 65% saturation with solid (NH4)2SO 4. The precipitate formed after 1 hr (stirring) was isolated by centrifugation (2800 rpm, 45 min). The supernatant was discarded and the pellet dissolved in the minimum volume of distilled water (21). (c) Acetone fractionation. This solution was cooled to - 2 ° C in a solid CO2/alcohol bath. Cold acetone was added over 40 min to give 30% v/v. The mixture was stirred for 30 min and centrifuged (2800 rpm, 30 min). To the supernatant cold acetone was added as above, to give 50% acetone (v/v). The pellet, from centrifugation, was suspended in distilled water (500 ml) and dialysed against water with several changes over 24 hr. The precipitate which formed was removed by centrifugation and the clear supernatant used. (d) Alcohol fractionation and heat denaturation. The enzyme was precipitated from this solution by slow addition of an equal volume of ethanol (95%) at - 6 ° C and centrifuged (2800 rpm, 60 min). The pellet was taken up in chilled distilled water (500 ml) and brought rapidly to 5ffC and maintained at this temperature for 5 min. The mixture was cooled, centrifuged and the bulky precipitate washed with distilled water (150ml) and the aqueous washings combined. The volume of the supernatant was reduced to 50 ml (Amicon, PM 10 membrane). (e) G- 75 gel permeation chromatography. Sephadex G-75, equilibrated with Tris HC1 buffer (25 mM, pH 7.50), was packed into a column (2.6 x 73 cm) at a linear flow rate of 6 0 m l h r t. The sample (10ml, l l 6 m g protein/ml) was applied and eluted with the above buffer at a linear flow rate of 27 ml hr -~. Active fractions were pooled and concentrated (Amicon, PM-10). (f) Chromatofocusing. Chromatofocusing, a relatively recent procedure, is a high resolution technique that recognizes the isoelectric point (pI) of a protein and employs a net anionic buffer exchange group immobilized on a highly cross-linked Sepharose CL-6B stabilizing medium and the formation of a pH gradient within the matrix by amphoteric buffering compounds. Polybuffer exchanger gel (20 ml, pH range 9~5) was equilibrated with Tris-CH3COOH buffer (25 mM, pH 8.30) and packed at a linear flow rate of 80 ml hr-~ (column, 1.0 x 25 cm). The column packing was checked using bovine cytochrome c (3 mg/ml, l ml), which is coloured, highly basic (pl = 10.50) and therefore repelled from the gel. The isoelectric point of glyoxalase I from yeast had been reported to be ~ 7 (MarmstS.1 et al., 1979) and so a pH gradient was designed to operate over the range 8~5. A solution of polybuffer 96 (diluted 1:13) was adjusted to the limit pH 6 with CH3COOH. Volumes required for the pH gradient were: 3 column volumes before gradient start and 9 column volumes for the gradient itself. Before sample application elution buffer (5 ml) was run onto the column. The sample (7 ml, 200 mg total) was then applied via a flow adaptor and syringe followed by elution buffer at a linear flow rate of 12 ml hr -~. Using this method the sample was not exposed to pH extrema. After elution the linearity of the pH gradient was checked and fractions assayed for enzyme activity.
(g) S-hexyl-glutathione 6B affinity chromatograph)'. Shexyl-glutathione was immobilized via the free amino function to epoxy-activated Sepharose 6B as described (Marmst~l et al., 1979). The gel was equilibrated with Tris HCI buffer (10mM, pH 7.80) and packed into an adjustable column (I .0 x 22 cm). Enzyme from the previous chromatofocusing step was loaded onto the column (typically 100ml, l - 2 m g m l i). The column was then washed with the same buffer supplemented with 0.2 M NaC1 (80 ml). The enzyme was then eluted (flow rate 3 0 m l h r -t) using S-hexyl-glutathione (3 mM) and glutathione (5 mM) in Tris-HCl buffer (10 mM, pH 7.80). The column was then re-equilibrated with starting buffer. (h) Gel permeation chromatograph)'. Sephadex G-100 SF, in a column (2.6× 70cm), equilibrated with Tris-HC1 (10mM, pH 7.80), was calibrated with molecular weight standards and enzyme samples (4% of the total bed volume) eluted (flow rate of 7mlhr-K). The enzyme was then ultrafiltered and concentrated. Aliquots (1.5 ml) of concentrated enzyme (3.5 mg ml-~) were stored frozen at - 2 f f ' C .
Molecular Weight Determination (a) Gel permeation chromatograph),. Molecular weights were determined by gel filtration on a G-100 SF Sephadex column (2.6 × 70cm) equilibrated with Tris HC1 buffer (10mM, pH 7.80). Standard proteins as well as uracil (10 and 2 mg/ml respectively) were run concurrently for column calibration. The molecular weights of the protein standards were: myoglobin, 17,800; trypsin inhibitor protein, 20,000; ovalbumin, 43,000; bovine serum albumin, 67,000 and catalase, 232,000 as void volume indicator. Blue dextran was not used as it has been reported (Kester, 1974) to bind to glyoxalase I. The column flow rates were checked before and after each run to ensure the validity of the standard curve. (b) Ultracentrifugation: sedimentation coefficient. Samples of yeast glyoxalase I were spun at 55,970 rpm, 20.05°C. Photographs were taken at 2 min intervals over 20 min. The sedimentation coefficient was calculated from a plot of In r vs time, where r is defined as the distance of the "'peak" in the Schlieren pattern from the axis of rotation. Protein concentrations were 1.7 and 3.5 mg/ml in 0.05 M potassium phosphate, pH 6.7, containing 0.1 M sodium chloride. The densities of the analysed solutions were measured as 0.9906 and 0.9968 g ml -t, respectively. (c) Equilibrium ultracentrifugation: Meniscus depletion method. Sedimentation equilibrium ultracentrifugation experiments were conducted by the Meniscus depletion method of Yphantis (1964), using 0.5 mg/ml of glyoxalase I in 0.05 M potassium phosphate buffer (pH 6.60) supplemented with 0.1 M sodium chloride; the solution density was 1.00 g ml -L. Centrifugation was carried out at 20.07°C (26 hr, 27998 rpm). The Rayleigh interferogram produced was analysed by a Joyce Loebl double-beam recording microdensitomer. An average of five analyses was used. The molecular weight was calculated from a plot of In C vs x 2, where C is the concentration of solute in arbitrary units and x is the radial distance. The slope of the line is dlnC dx2 • The partial specific volume for glyoxalase 1 (~) was calculated (Schachman, 1957) from the partial specific volumes of the component amino-acids, giving a value of 0.73 cm 3 g-i. RESULTS
(a) Isolation o f Glyoxalase I from Yeast Table 1 s u m m a r i z e s the purification d a t a for yeast glyoxalase I. T h e first c h r o m a t o g r a p h y step (Sephadex G-75, p H 7 . 8 0 ) r e m o v e d ~ 7 5 % o f cont a m i n a t i n g p r o t e i n material. This was f o l l o w e d by
551
Yeast glyoxalase I Table 1. Purification of glyoxalase I from yeast Vol (ml)
Total protein (rag)
1.65 × 104
8.91 x 105
3.3 × 103
3.35 x 105
2.0 × 103
4.40 × 104
50 500 520 36 28
Step Chloroform extraction 4(~65% (NH4)2SO4 fractionation 33 50% Acetone fractionation + dialysis 50% Ethanol fractionation/ heat denaturation (PM 10 Amicon concentrate) Sephadex G-75 Chromatofocussing S-Hexylglutathione Sepharose 6B Sephadex G-100 SF
Specific activity (Fmol/min/mg)
Total activity (#mol/min)
Yield (%)
Purification factor
1.80
1.604 × 106
100.0
"'1.00'"
4.38
1.47 x 106
92.0
2.44
17.1
7.53 × 105
47.0
9.50
5.8 × 103 1.45 × 103 3.02 x 102
57.8 141.4 496.7
3.35 × 105 2.05 × 105 1.50 x 105
21.0 12.80 9.35
32.1 78.6 276.0
1.07 x 102 96.00
1028.00 1042.00
1.10 x 105 1.01 x 105
6.85 6.30
571.0 580.0
" c h r o m a t o f o c u s i n g " . F i g u r e 1, o f the p H , A280 a n d e n z y m e activity profiles for this step, s h o w s e n z y m e activity a s s o c i a t e d with 4 p r o t e i n p e a k s at p H values 7.90, 7.70, 7.50 a n d 7.10, called p e a k s I, II, III a n d IV respectively. A n a l y s e s o f these p e a k s by D I S C 10% a n o d i c a n d S D S - 1 0 % P A G E are s h o w n diag r a m a t i c a l l y in Fig. 2. T h e D I S C r o d gels o f each p e a k s h o w e d multiple b a n d s , w h e r e a s 1 0 % - P A G E S D S r o d gels s h o w e d 3 clear b a n d s . C o m p a r i s o n o f the S D S gels with a G-75 s a m p l e revealed s o m e
\
\
\
c o m m o n b a n d s , t w o over a m o l e c u l a r weight range o f 43,000 a n d 68,000 a n d o n e e s t i m a t e d b e t w e e n 30,000 a n d 35,000. P e a k s I a n d II were t h e n t r e a t e d s e p a r a t e l y in s u b s e q u e n t purification steps; p e a k s III a n d IV were c o m b i n e d a n d f u r t h e r purified. T h e c h r o m a t o f o c u s i n g d a t a in the purification table refers to p e a k s I a n d II since the a m o u n t o f material p r e s e n t in p e a k s III a n d IV was small. Affinity c h r o m a t o g r a p h y using S - h e x y l g l u t a t h i o n e as the i m m o b i l i z e d ligand fulfilled t w o f u n c t i o n s in
\
2-
\
\ \ I
\
\
\
IE u
o
\
"1" ~X
ea
A
-
~ . .
2
4.......___
4
6
8
10
.
12
.
.
.
14
16
Time (hr)
Fig. 1. Chromatofocussing of yeast glyoxalase I. The elution profile shows the fractionation of pooled G-25 material applied to a column (1.0 x 25 cm) containing PBE 96 gel, equilibrated with Tris42H3COOH buffer (25 mM, pH 8.4). Protein was eluted using polybuffer 96 (1:13 dilution factor) adjusted to the limit pH 6.00 with CH3COOH, at a linear flow rate of 12 ml hr -~. - - - , A280;- - . - - , glyoxalase I activity; . . . . . , pH profile.
552
KENNETH T. DOUGLAS et al.
+
Table 2. Carbohydrate composition of yeast glyoxalase I Weight %
I
II
III
IV
Fucose Mannose Galactose Glucose Mannosamine Galactosamine Glucosamine N-Acetyl-neuraminic acid
0.15 0.17 0.16 0.03 0.06 0.17 -0.01 Total
0.75
molecule behaves ideally following a continuous flow pattern and that the shape is of a spherical nature.
I
II
III
IV
Fig. 2. Upper: DISC-PAGE of chromatofocussing peaks I, II, III and IV on 10% polyacrylamide rod gels. Lower: SDS-PAGE of peaks I, II, III and IV on 10% polyacrylamide rod gels. Molecular weights of components present in these peaks were estimated by comparison of standard molecular weight proteins where: 1, bovine serum albumin, 67,000; 2, ovalbumin, 43,000; 3, myoglobin, 17,200; 4, cytochrome c, 11,500. the next step: (I) removal of polybuffer components from the previous step and (2) efficient removal of contaminating proteins utilizing the enzyme's binding activity. The preparation after G-100 SF gel filtration was homogeneous when judged as a single band stained with Coomassie Blue on 15% homogeneous PAGE and 10-18% gradient SDS-PAGE under both normal and overloading sample concentrations. Anodic DISC PAGE on 10% acrylamide slab gels showed a single component, but on rod gels showed two, tight bands. However, on incubation with 100mM GSH (pH 7.0) only one band was detected. Isoelectric points were determined by means of thin layer polyacrylamide plates using standard protein pl markers. The glyoxalase I sample showed 4 major bands at pI's: 6.60, 7.10, 7.25 and 7.75 with two faint bands at 6.80 and 7.65. The number of bands was not reduced by prior incubation with GSH (100mM, pH7.0), KCN (10mM), DTT (50mM). G-100 purified samples of peaks I and II obtained from the chromatography step were identical by this criterion. (b) Ultracentrt[uge data The data obtained for the estimation of S (sedimentation velocity) showed a single peak in the Schlieren pattern and the plot of In r vs time was linear. Data from molecular weight estimation by the sedimentation equilibrium Meniscus depletion method of Yphantis (1964) yielded a linear plot of In C vs (x) 2. Using the appropriate formulae (Bowen and Rowe, 1970), the diffusion coefficient calculated for glyoxalase I was 9.534 x 10 7cm2/sec, with a calculated frictional ratio J'~['o= 1.03, indicating that the
(c) Amino-acid and carbohydrate composition The amino-acid composition of yeast glyoxalase 1 (24, 48 and 72hr hydrolyses) was close to that reported by Marmst~.l and Mannervik, 1978, the main differences being in the Asp, Thr, Ser and Phe contents. In agreement with these authors we also observed routinely 30-33 tryptic peptides on 2-dimensional mapping, in line with a monomeric structure. The total carbohydrate content based on a molecular weight of 3.4 x 104 was 0.75% with fucose, mannose, galactose and galactosamine accounting for 0.65 wt% (see Table 2). (d) Molecular weight of glyoxalase I from yeast The molecular weight of yeast glyoxalase I, determined by SDS-PAGE, was 37,000 from homogeneous 15% PAGE and 35,500 from gradient !(~18% PAGE. The molecular weight was also estimated as 33,800 + 3,300 by gel permeation chromatography on Sephadex G-100 SF (average of 5 samples). In sedimentation velocity experiments a sedimentation coefficient of 3.74+0.115 was obtained. A value of 3.60 ($20,,,.) for the yeast enzyme (Aronsson and Mannervik, 1977) has been reported. The weightaverage molecular weight of yeast glyoxalase I estimated by the Meniscus depletion method of equilibrium ultracentrifugation was found to be 35,800 ___220.
(e) Kinetic parameters With the methylglyoxal/glutathione hemithioacetal as substrate, the following values were obtained at pH6.60, 25°C: kc,t=3.54x 104min i. K,,=5.12_+ 0.33× 10 4M, k , ~ / K , , = 6 . 9 x 107M ~min-'. The values of these parameters are similar to those in other reports in the literature (Aronsson and Mannervik, 1977; Van der Jagt and Hart, 1973). The magnitude ofkcat/K m (2.30 × 107 M -l sec ') above is based on a K,, value calculated from the total concentration hemimercaptal present. As glyoxalase I has been shown under some conditions to use only one of the diastereomeric forms of this hemimercaptal, the actual value of k~,t/K,,, may be greater (~4.6 x 107 M -I sec t). In either case glyoxalase I is one of the most catalytically active of enzymes with a k ~ S K m value close to that set by the diffusion-limit (Fersht, 1977).
Yeast glyoxalase I
(A)
553 e
(B)
I
A I
52
4
%2
7
% b.q
<] 2 2 I
L
n
J
I
200
I
250
250 Wavelength
Wavelength
(nm)
300 (nm)
A
'E u (Cl
7 1.o o c:: ',v 0.8 ,,~ 0.6 I ~ 0.4 4~
0.2 £a
?,
\.
•
I
I
I
250 Wavelength
:k ,
- ~qro.4~-...I 300
(nm)
Fig. 3. (A) Near- and (B) far-UV CD spectra of yeast glyoxalase l (0.5 mg/ml 0.05 M KH2PO4 buffer, pH6.80 and 0.08% v/v, dmso as co-solvent at 23.5°C) in the presence and absence of S-(4bromobenzyl)glutathione (5.2 x l0 5 M, I). Near-UV and far-UV spectra were recorded in duplicate in 0.2 and l0 mm pathlength cells, respectively. There was no significant spectral shift in the far-UV region in the presence of inhibitor. (C) Aromatic CD difference spectrum of yeast glyoxalase I [+ and S-(4-bromobenzyl)glutathione]. -
(f) Circular dichroic (CD ) spectra of yeast glyoxalase I The CD spectra shown in Fig. 3 were recorded in the presence and absence of the strong competitive inhibitor, S-(4-bromobenzyl)-glutathione. The binding of the competitive inhibitor has no major effect on the global secondary structure (considering the region below 220 nm). For the native protein no one conformation (~, fl or random coil) dominates. The (240-300 nm) CD spectrum of yeast glyoxalase I exhibits bands at 264, 281 and 293 nm which are almost certainly attributable to phenylalanine, tryptophan and tyrosine. CD band positions and intensities in this region were found to be temperature independent over the range 23.5-43°C. However, binding of S-(4-bromobenzyl)-glutathione caused small changes in C D intensities with a slight red shift in the 264 nm band and a blue shift in the 293 nm band. The CD difference spectrum (Fig. 3C) has given rise to a positive maximum at 275 rim. The overall shape is similar to CD spectra of tyrosine model compounds (Strickland et al., 1970). The difference spectrum reflects perturbations coincident with the binding of competitive inhibitor. The location of the 275 nm CD maximum leads us to tentatively interpret it as reflecting a tyrosine perturbation, but insufficient fine structure is present to
allow unambiguous assignment. The spectrum possesses no maxima >286 nm, therefore, tryptophan perturbations, if present, are not identifiable. (g) Dependence of K i values The inhibition of yeast glyoxalase I by S-(pbromobenzyl)glutathione has been established to be competitive (Marmst~l and Mannervik, 1979; Douglas et al., 1982) as it has for S-(pazidophenacyl)glutathione (Seddon and Douglas, 1980). For the former Ki was essentially pH independent in the range 5.80-8.00 (Douglas and Ghobt-Sharif, in preparation). For S-(pbromobenzyl)glutathione the mean Ki value of the pH range 5.80-8.00 was found to be 3.41 _ 0.80/aM. For S(p-azidophenacyl)glutathione Ki values were as follow (pH in parentheses): 0 . 7 0 + 0 . 0 3 x 10-4M (5.50), 1.05 _ 0.05 x 10 -4 M (6.60), 1.08 _ 0.08 x 10-4M (7.52): mean value 0.94+__0.21 × 10-4M. DISCUSSION
The purification procedure described gives an apparently homogeneous enzyme from yeast, as judged by SDS, DISC PAGE, chromatographic, ultracentrifugal analysis criteria. The enzyme was purified about 600-fold in a yield of 6.3% to a specific activity
554
KENNETH T. DOUGLASet al.
of 1042 units/mg of protein and was stable when stored at - 20°C. The specific activity achieved agrees well with literature values (Davis and Williams, 1966; Van der Jagt and Han, 1973; Marmst~l and Mannervik, 1978). The molecular weight of the enzyme, by gel permeation chromatography and equilibrium centrifuge analysis, was found to be 33,800 + 3,300 and 35,800_+ 220, respectively. These values are in good internal agreement and with literature estimates (from gel filtration and/or sedimentation velocity estimates) of 32-35,000 (Van der Jagt and Han, 1973; Mannervik et al., 1972; Marmst~l and Mannervik, 1978). The presently-determined sedimentation coefficient (3.74 s) and the more or less spherical shape of the protein molecule, indicated by the close to unity value of the frictional ratio ( f i f o = 1.03) is in agreement with the results of another study (Aronsson and Mannervik, 1977). The isoelectric point (pI) of yeast glyoxalase I has been estimated (Marmsffd et al., 1979) as ~ 7 from an isoelectric focusing column (eluted fractions were assayed for glyoxalase I activity which came out in 43 ml of a 110 ml total column eluant; pH gradient 8 5). Clearly, the resolution/precision of such a procedure is low. Analytical electrofocusing on gels of the present preparation gave 4 major protein bands (pI's 6.60, 7.10, 7.25 and 7.75). Several isoelectrophoretic forms have been reported for glyoxalase I isolated from mammalian sources (Marmst~.l et al., 1979; Uotila and Koivusalo, 1975, 1980). This apparent heterogeneity was lifted by prior treatment with GSH in many cases, but this had no effect on the electrophoretic results given by fresh haemolysates (Uotila and Koivusalo, 1980) and this yeast preparation. These multiple bands may represent true heterogeneity or may be artefactual. Aronsson and Mannervik (1977) have suggested these isoelectrophoretic forms may originate from a mixed disulphide of the enzyme and glutathione. However, while the yeast enzyme has accessible SH groups, the mammalian enzymes (also with multiple bands on electrofocussing) do not (Ekwall and Mannervik, 1970; Mannervik et al., 1975). Glycoproteins may exhibit multiple bands if the sugar residues present are inhomogeneous. In the present study yeast glyoxalase I on carbohydrate analysis revealed a small, but significant carbohydrate content (0.75% by weight) representing an average of 2 sugar units per enzyme molecule. Mannose and galactosamine are typically found associated with yeast proteins, linked via an O-glycosidic linkage through the hydroxyl group of serine or threonine (Gottschalk, 1972; Sharon, 1975). The presence of a carbohydrate component in yeast glyoxalase I has not previously been reported. The multiple bands on electrofocussing of yeast glyoxalase I may arise in part from this polydispersity caused by variable sugar contents of individual molecules. The carbohydrate analysis (Table 2) reveals 7 difJerent types of sugar (the small amount of glucose present may be derived from the final Sephadex G-100 step). As there are on average only 2 sugar residues per protein molecule, the preparation must by polydisperse in this respect of sugar content. Clearly from the data in this work [for S-(p-azidophenacyl)glutathione and S-(p-bromoben-
zyl)glutathione] as well as that reported for S-(mtrifluoromethylbenzyl)glutathione and N-acetyI-S(p-bromobenzyl)glutathione (Douglas et al., 1982a) there is little or no effect of pH on the K, values for competitive inhibitors possessing the glutathione backbone structure. This stands in marked contrast to the pH dependence of Km reported for glutathione hemimercaptals formed with methylglyoxal, phenylglyoxal and p-chlorophenylglyoxal (Van der Jagt and Han, 1973). The magnitude of Km depends on two ionisations of pKapv values 4.7-5.2 and 8.4-8.9, depending on the substrate. For all three substrates Vmax was pH-insensitive, which was suggested to indicate that in this pH range no ionisable groups occur which are important in the conversion of substrate to product. The groups observed for K,, were ascribed to enzymatic ionisations rather than dissociations associated with glutathione, with the group at pKapp ~ 5 ascribed to a ~ O 2 H site and that at ~ 8.7 to an - - N H + group (Vander Jagt and Han, 1973). An apparent related anomaly for human erythrocyte glyoxalase I is that the K,, value for the adduct of methylglyoxal and glutathione was effectively unchanged by substitution of Co 2+, Mn 2+ or Mg 2+ for the crucial Zn z+ of the native enzyme, whilst the competitive inhibition constant (K~) for S-(pbromobenzyl)glutathione was sensitive to the nature of the metal ion (Aronsson et al., 1981). Consider the simple scheme for enzyme catalysis of equation (2) for which K m = ( k 2 + k 3 ) / k ~ and K s = k2/k I (the enzyme-substrate complex dissociation constant). kl
k3
E + S~ES--* E + P
(2)
k2
For competitive inhibition, we also have equation (3) with K~ = ks~k4. k4
E + I~-~--EI k5
(3)
If S and I bind to the same site then clearly K,, --~ K~ for this enzyme as both I and S would have similar pH dependencies, which is not observed. Nor is this model possible with K,, = (k: + k3)/k~ with the pH dependence of Km ascribed to the k 3 term (not present in Ki) as Vm,x(=k3[E0]) has no pH-dependence. If, as Vander Jagt and Han (1973) argue, K,, = k3/k ~, a problem arises in the pH dependence unless k~ and k2 have equal but opposite pH dependences which cancel (as k4 and k 5 pH dependencies must have balanced each other for K0. The dispersion in metal ion sensitivity of Km and K, may arise if the metal ion does not lie in the activesite, but there are good circumstantial arguments against this (Aronsson et al., 1981). Alternatively Ki may be wrongly defined for glutathione analogues. The equilibrium binding constant of S-(pbromobenzyl)glutathione is not significantly different from its steady-state inhibition constant (l/K,) (Marmst~.i and Mannervik, 1979). However, although glutathione does not affect the equilibrium binding of S-(p-bromobenzyl)glutathione it competes directly with this S-blocked analogue under turnover conditions. This was explained by Marmsthl and Mannervik (1979) by suggesting that glyoxalase I exists in an "equilibrium" form which does not bind
Yeast glyoxalase I glutathione and a "catalytic" conformation which does. The C D spectrum of glyoxalase I is slightly sensitive to S-(p-bromobenzyl)glutathione in the aromatic region and it is possible from the results that binding of this inhibitor perturbs the environment of a tyrosine residue. This tends to support the view of Marmsffd and Mannervik with the different enzyme conformations being inhibitor inducible although a minor configurational change is all that is necessary to explain the C D results. SUMMARY
Glyoxalase I (EC 4.4.1.5) has been purified ~600-fold from baker's yeast by a procedure involving chromatofocussing and affinity chromatography. The enzyme contained 0.75% w/w carbohydrate consisting of fucose, mannose, galactose, mannosamine, galactosamine, N-acetylneuraminic acid and possibly glucose. The molecular weight was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis to be ~36,000, 33,800 + 3300 by gel filtration and 3 5 , 8 0 0 _ 200 by equilibrium centrifugation. The sedimentation coefficient was 3.74 s. F o u r isoelectric forms were detected with pI values of 6.60, 7.10, 7.25 and 7.75, probably because of the polydispersity arising from the sugar component. The circular dichroic spectra were measured in the absence and presence of the competitive inhibitor S-(p-bromobenzyl)glutathione, the presence of which appeared to perturb a tyrosine residue. The effect of pH on Ki for this inhibitor was measured and pH effects on glyoxalase I discussed. Acknowledgements--We are grateful to the Medical Re-
search Council for support (A.P.S.) and to Drs R. C. Hider and A. Drake for assistance in the CD aspects. REFERENCES
Alexander N. M. and Boyer J. L. (1971) A rapid assay for the glyoxalase enzyme system. Analyt. Biochem. 41, 29-35. Aronsson A.-C. and Mannervik B. (1977) Characterisation of glyoxalase I purified from pig erytbrocytes by affinity chromatography. Biochem. J. 165, 503-509. Bergmeyer H. U. (1974) Methods of Enzymatic Analysis, 2nd edn (Eng.), pp. 283-284. Academic Press, New York. Bowen T. J. and Rowe A. J. (1970) Introduction to Ultracentrifugation, pp. 85-96. Wiley, New York. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analyt Biochem. 72, 248-255. Douglas K. T., AI-Timari A., D'Silva C. and Gohel D. I. (1982a) Role of the N-terminus of glutathione in action of yeast glyoxalase I. Biochem. J. 207, 323-332. Douglas K. T., Nadvi I. N. and Ghotb-Sharif J. (1982b) Porphyrin inhibition of glyoxalase I. IRCS med. Sci. 10, 861-862. Ekwall K. and Mannervik B. (1970) Inhibition of yeast S-lactylglutathione lyase (glyoxalase I) by sulfhydryl reagents. Archs Biochem. Biophys. 137, 128-132.
555
Fersht A. (1977) Enzyme Structure and Mechanism, p. 132. Freeman, San Francisco. Gillespie E. (1978) Concanavilin A increases glyoxalase enzyme activities in polymorphonuclear leukocytes and lymphocytes. J. lmmunol. 121, 923-925. Gillespie E. (1979) Effects of S-lactoylglutathione and inhibitors of glyoxalase 1 on histamine release from human leukocytes. Nature 277, 135-137. Gottshalk A. (1972) Glycoproteins 5A, 470-476. Han L. P. B. (1975) Ph.D. Thesis, University of New Mexico, p. 20. lio M., Okabe K. and Omura H. (1976) Effect of reductones on glyoxalase I. J. nutr. Sci. Vitaminol. 22, 53 61. Kester M. V. (1974) Ph.D. Thesis, North Texas State University, p. 47. Mannervik B., Lindstrom L. and Bartfai T. (1972) Partial purification and characterisation of glyoxalase I from porcine erythrocytes. Eur. J. Biochem. 29, 276-281. Mannervik B., Ekwall K., Marmst~l E. and Gorna-Hall B. (1975) Inactivation of glyoxalase I from porcine erythrocytes and yeast by amino-group reagents. IBM., 53, 327 333. Margolis H. C., Nakagawa Y., Douglas K. T. and Kaiser E. T. (1978) Multiple forms of carboxypeptidase Y from Saccharomyces cerevisiae. J. biol. Chem. 253, 7891 7897. Marmsffd E. and Mannervik B. (1978) Subunit structure of glyoxalase I from yeast FEBS Lett. 85, 275-277. Marmstgd E. and Mannervik B. (1979) Binding of the competitive inhibitor S-(p-bromobenzyl)glutathione to glyoxalase I from yeast. FEBS Lett. 102, 162-164. MarmstS.1 E., Aronsson A.-C. and Mannervik B. (1979) Comparison of glyoxalase I purified from yeast (Saccharomyees cerevisiae) with enzyme from mammalian sources. Biochem. J. 183, 23-30. Racker E. (1951) The mechanism of action of glyoxalase. J. biol. Chem. 190, 685-696. Schachman H. K. (1957) Ultracentrifugation, diffusion and viscometry. Meth. Enzymol. 4, 32 et seq. Seddon A. P. and Douglas K. T. (1980) A photoaffinity label derivative of glutathione and its inhibition of glyoxalase I. FEBS Lett. 110, 262-264. Sharon N. (1975) Complex Carbohydrates, Their Chemistry, Biosynthesis and Functions. Addison-Wesley, Massachusetts. Spector S. S. (1978) Refinement of the Coomassie Blue method of protein determination. Analyt. Biochem. 86, 142-146. Strickland E. H., Wilchek M., Horwitz J. and Billups C. (1970) Low temperature circular dichroism of tyrosyl and tryptophanyl diketopiperazines. J. biol. Chem. 245, 4168-4177. Szent-Gyorgi A. (1968) Bioelectronics. Science 161, 988-990. Uotila L. and Koivusalo M. (1975) Purification and properties of glyoxalase I from sheep liver. Eur. J. Biochem. 52, 493-503. Uotila L. and Koivusalo M. (1980) Separation of the isoenzymes of glyoxalase I from human red blood cells by electrophoresis and isoelectric focussing on polyacrylamide gel and by ion exchange chromatography. Acta chem. scand. B34, 63-68. Van der Jagt D. L. and Han L.-P. B. (1973) Deuterium isotope effects and chemically modified coenzymes as mechanism probes of yeast glyoxalase I. Biochemistry 12, 5161-5167. Vince R. and Daluge S. (1971) Glyoxalase I inhibition. A possible approach to anticancer agents. J. reed. Chem. 14, 35-37. Yphantis D. A. (1964) Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3, 297-317.
Int. J. Biochem. Vol. 18, No. 6, pp. 549 555, 1986
Printed in Great Britain. All rights reserved
CIRCULAR
Y E A S T G L Y O X A L A S E I. D I C H R O I C S P E C T R A A N D pH EFFECTS
KENNETH T. DOUGLASI*, ANDREW P. SEDDONtt and YASHUSHI NAKAGAWA2 IDepartment of Chemistry, University of Essex, Colchester, Essex CO4 3SQ, U.K. [Tel. (0206) 862286] 2Michael Reese Hospital and Medical Center, 29th Street and Ellis Avenue, Chicago, IL 60616, U.S.A. (Received 31 October 1985) AMtraet--l. Large scale isolation and physicochemical characterisation of yeast glyoxalase I showed that this enzyme contained small amounts of carbohydrates. 2. Circular dichroic spectra of the enzyme measured in the presence and absence of S-(pbromobenzyl)glutathione indicated perturbation of a tyrosine on binding of this competitive inhibitor. 3. Values of K, for competitive inhibitors were pH invariant over the accessible pH range.
inhibition of glyoxalase I by porphyrins (Douglas et al., 1982b). This presumably implies a conformational difference in the forms of enzyme which bind substrate and inhibitor respectively. Considerable evidence of flexibility in this system has been noted (Douglas and Shinkai, 1985).
INTRODUCTION
Glyoxalase I (EC 4.4.1.5) converts the hemithiolacetal formed between glutathione (GSH) and methylglyoxal or other ~-ketoaldehydes into Slactoylglutathione etc. (Racker, 1951), which is then hydrolysed under glyoxalase II catalysis, see equation (1).
GS GSH + CH3COCHO
I
C-----O
GS
I
~-- H - - C - - - O H
I
glyoxalase I
, H---C --OH [ CH 3
C CH 3
0
GSH glyoxalas¢ , + I1
(1)
COl H-@--OH CH3
A variety of suggestions concerning the function of glyoxalase I has been made ranging from roles in cell proliferation/cancer (Szent-Gyorgi, 1968), enhancement of anti-IgE-induced histamine release (Gillespie, 1979) and microtubule assembly (Gillespie, 1975) to protection against intestinal bacteria (Aronsson et al., 1977). Our interest in this enzyme stems from many reports that glyoxalase I inhibitors often have anticancer activity (Vince and Daluge, 1971; Iio et al., 1975). The pH profile for the yeast glyoxalase I-catalysed reaction of a n u m b e r of ct-ketoaldehydes has been reported (Van der Jagt and Han, 1973). It was found that /)max was insensitive to pH, while K,~ values increased at lower and higher pH (with pK~pp values of 4.7-5.2 and 8.4-8.9, depending on the ~t-ketoaldehyde). In spite of the pH-dependence of K,, we have found that competitive inhibition constants for S-blocked and for S,N-blocked glutathiones are essentially pH-independent (Douglas et al., 1982a) in marked contrast to the pH-dependent competitive
MATERIALS AND METHODS
*Author to whom correspondence should be addressed. tPresent address: Department of Biochemistry, Cornell University Medical College, New York, NY 10021, U.S.A.
The following materials were obtained from Sigma Chemical Co. Ltd. (Dorset, England): methylglyoxal (40% aqueous solution), reduced glutathione, calibration proteins for SDS molecular weight determinations, semicarbazide hydrochloride. Tetranitromethane was from Aldrich Chemical Co. Ltd. Other commercial materials were of the highest quality available. Methylglyoxal was purified according to Han (1975) and standardized by a modification of the enzymatic method (Bergmeyer, 1974) and also by a semicarbazide procedure (Alexander and Boyer, 1971). The glyoxalase I assay system (after Racker, 1951) contained GSH (0.66mM), methylglyoxal (2mM) and KH2PO 4 (0.05 M, pH 6.60) in a final volume of 3.0 ml. This mixture was incubated at 25°C (5 min) to ensure complete equilibration to the hemimercaptal. Reaction was initiated by addition of glyoxalase I and the absorbance at 240 nm recorded against time using a Pye-Unicam SP8100 UV-visible spectrophotometer at 25°C. A unit of glyoxalase I is here defined as the amount of enzyme catalysing formation of 1 #mol of S-o-lactoylglutathione per minute under the above conditions. Protein concentrations were determined spectrophotometrically using a dye-binding procedure (Bradford, 1976; Spector, 1978) for more purified samples. Purification of Glyoxalase I from Yeast (a) Autolysis. Compressed baker's yeast (20 kg), crumbled and mixed with chloroform (41), was left with occasional
qztO
550
KENNETH T. DOUGLAS et al.
stirring for 1 hr or until the mixture liquefied. Distilled water (81) was added, the pH was adjusted to 6.6 with sodium hydroxide (I M) and readjusted after 2 hr and this suspension was left overnight at room temperature. All subsequent steps were performed at 4'C unless otherwise stated. The autolysed suspension was centrifuged (2800rpm, 40mins, MSE "Mistral 4 1" centrifuge, Head No. 43124-105). The heavy precipitate was discarded. (b) Ammonium sulphate fractionation. The supernatant was fractionated over a 40-65% (NH4)2SO 4 range. Firstly, (NH4)2SO 4 was added to the supernatant to give 40% saturation. The solution was stirred for ~ 1 hr and centrifuged (2800 rpm, 40 min). The precipitate was discarded and the supernatant brought to 65% saturation with solid (NH4)2SO 4. The precipitate formed after 1 hr (stirring) was isolated by centrifugation (2800 rpm, 45 min). The supernatant was discarded and the pellet dissolved in the minimum volume of distilled water (21). (c) Acetone fractionation. This solution was cooled to - 2 ° C in a solid CO2/alcohol bath. Cold acetone was added over 40 min to give 30% v/v. The mixture was stirred for 30 min and centrifuged (2800 rpm, 30 min). To the supernatant cold acetone was added as above, to give 50% acetone (v/v). The pellet, from centrifugation, was suspended in distilled water (500 ml) and dialysed against water with several changes over 24 hr. The precipitate which formed was removed by centrifugation and the clear supernatant used. (d) Alcohol fractionation and heat denaturation. The enzyme was precipitated from this solution by slow addition of an equal volume of ethanol (95%) at - 6 ° C and centrifuged (2800 rpm, 60 min). The pellet was taken up in chilled distilled water (500 ml) and brought rapidly to 5ffC and maintained at this temperature for 5 min. The mixture was cooled, centrifuged and the bulky precipitate washed with distilled water (150ml) and the aqueous washings combined. The volume of the supernatant was reduced to 50 ml (Amicon, PM 10 membrane). (e) G- 75 gel permeation chromatography. Sephadex G-75, equilibrated with Tris HC1 buffer (25 mM, pH 7.50), was packed into a column (2.6 x 73 cm) at a linear flow rate of 6 0 m l h r t. The sample (10ml, l l 6 m g protein/ml) was applied and eluted with the above buffer at a linear flow rate of 27 ml hr -~. Active fractions were pooled and concentrated (Amicon, PM-10). (f) Chromatofocusing. Chromatofocusing, a relatively recent procedure, is a high resolution technique that recognizes the isoelectric point (pI) of a protein and employs a net anionic buffer exchange group immobilized on a highly cross-linked Sepharose CL-6B stabilizing medium and the formation of a pH gradient within the matrix by amphoteric buffering compounds. Polybuffer exchanger gel (20 ml, pH range 9~5) was equilibrated with Tris-CH3COOH buffer (25 mM, pH 8.30) and packed at a linear flow rate of 80 ml hr-~ (column, 1.0 x 25 cm). The column packing was checked using bovine cytochrome c (3 mg/ml, l ml), which is coloured, highly basic (pl = 10.50) and therefore repelled from the gel. The isoelectric point of glyoxalase I from yeast had been reported to be ~ 7 (MarmstS.1 et al., 1979) and so a pH gradient was designed to operate over the range 8~5. A solution of polybuffer 96 (diluted 1:13) was adjusted to the limit pH 6 with CH3COOH. Volumes required for the pH gradient were: 3 column volumes before gradient start and 9 column volumes for the gradient itself. Before sample application elution buffer (5 ml) was run onto the column. The sample (7 ml, 200 mg total) was then applied via a flow adaptor and syringe followed by elution buffer at a linear flow rate of 12 ml hr -~. Using this method the sample was not exposed to pH extrema. After elution the linearity of the pH gradient was checked and fractions assayed for enzyme activity.
(g) S-hexyl-glutathione 6B affinity chromatograph)'. Shexyl-glutathione was immobilized via the free amino function to epoxy-activated Sepharose 6B as described (Marmst~l et al., 1979). The gel was equilibrated with Tris HCI buffer (10mM, pH 7.80) and packed into an adjustable column (I .0 x 22 cm). Enzyme from the previous chromatofocusing step was loaded onto the column (typically 100ml, l - 2 m g m l i). The column was then washed with the same buffer supplemented with 0.2 M NaC1 (80 ml). The enzyme was then eluted (flow rate 3 0 m l h r -t) using S-hexyl-glutathione (3 mM) and glutathione (5 mM) in Tris-HCl buffer (10 mM, pH 7.80). The column was then re-equilibrated with starting buffer. (h) Gel permeation chromatograph)'. Sephadex G-100 SF, in a column (2.6× 70cm), equilibrated with Tris-HC1 (10mM, pH 7.80), was calibrated with molecular weight standards and enzyme samples (4% of the total bed volume) eluted (flow rate of 7mlhr-K). The enzyme was then ultrafiltered and concentrated. Aliquots (1.5 ml) of concentrated enzyme (3.5 mg ml-~) were stored frozen at - 2 f f ' C .
Molecular Weight Determination (a) Gel permeation chromatograph),. Molecular weights were determined by gel filtration on a G-100 SF Sephadex column (2.6 × 70cm) equilibrated with Tris HC1 buffer (10mM, pH 7.80). Standard proteins as well as uracil (10 and 2 mg/ml respectively) were run concurrently for column calibration. The molecular weights of the protein standards were: myoglobin, 17,800; trypsin inhibitor protein, 20,000; ovalbumin, 43,000; bovine serum albumin, 67,000 and catalase, 232,000 as void volume indicator. Blue dextran was not used as it has been reported (Kester, 1974) to bind to glyoxalase I. The column flow rates were checked before and after each run to ensure the validity of the standard curve. (b) Ultracentrifugation: sedimentation coefficient. Samples of yeast glyoxalase I were spun at 55,970 rpm, 20.05°C. Photographs were taken at 2 min intervals over 20 min. The sedimentation coefficient was calculated from a plot of In r vs time, where r is defined as the distance of the "'peak" in the Schlieren pattern from the axis of rotation. Protein concentrations were 1.7 and 3.5 mg/ml in 0.05 M potassium phosphate, pH 6.7, containing 0.1 M sodium chloride. The densities of the analysed solutions were measured as 0.9906 and 0.9968 g ml -t, respectively. (c) Equilibrium ultracentrifugation: Meniscus depletion method. Sedimentation equilibrium ultracentrifugation experiments were conducted by the Meniscus depletion method of Yphantis (1964), using 0.5 mg/ml of glyoxalase I in 0.05 M potassium phosphate buffer (pH 6.60) supplemented with 0.1 M sodium chloride; the solution density was 1.00 g ml -L. Centrifugation was carried out at 20.07°C (26 hr, 27998 rpm). The Rayleigh interferogram produced was analysed by a Joyce Loebl double-beam recording microdensitomer. An average of five analyses was used. The molecular weight was calculated from a plot of In C vs x 2, where C is the concentration of solute in arbitrary units and x is the radial distance. The slope of the line is dlnC dx2 • The partial specific volume for glyoxalase 1 (~) was calculated (Schachman, 1957) from the partial specific volumes of the component amino-acids, giving a value of 0.73 cm 3 g-i. RESULTS
(a) Isolation o f Glyoxalase I from Yeast Table 1 s u m m a r i z e s the purification d a t a for yeast glyoxalase I. T h e first c h r o m a t o g r a p h y step (Sephadex G-75, p H 7 . 8 0 ) r e m o v e d ~ 7 5 % o f cont a m i n a t i n g p r o t e i n material. This was f o l l o w e d by
551
Yeast glyoxalase I Table 1. Purification of glyoxalase I from yeast Vol (ml)
Total protein (rag)
1.65 × 104
8.91 x 105
3.3 × 103
3.35 x 105
2.0 × 103
4.40 × 104
50 500 520 36 28
Step Chloroform extraction 4(~65% (NH4)2SO4 fractionation 33 50% Acetone fractionation + dialysis 50% Ethanol fractionation/ heat denaturation (PM 10 Amicon concentrate) Sephadex G-75 Chromatofocussing S-Hexylglutathione Sepharose 6B Sephadex G-100 SF
Specific activity (Fmol/min/mg)
Total activity (#mol/min)
Yield (%)
Purification factor
1.80
1.604 × 106
100.0
"'1.00'"
4.38
1.47 x 106
92.0
2.44
17.1
7.53 × 105
47.0
9.50
5.8 × 103 1.45 × 103 3.02 x 102
57.8 141.4 496.7
3.35 × 105 2.05 × 105 1.50 x 105
21.0 12.80 9.35
32.1 78.6 276.0
1.07 x 102 96.00
1028.00 1042.00
1.10 x 105 1.01 x 105
6.85 6.30
571.0 580.0
" c h r o m a t o f o c u s i n g " . F i g u r e 1, o f the p H , A280 a n d e n z y m e activity profiles for this step, s h o w s e n z y m e activity a s s o c i a t e d with 4 p r o t e i n p e a k s at p H values 7.90, 7.70, 7.50 a n d 7.10, called p e a k s I, II, III a n d IV respectively. A n a l y s e s o f these p e a k s by D I S C 10% a n o d i c a n d S D S - 1 0 % P A G E are s h o w n diag r a m a t i c a l l y in Fig. 2. T h e D I S C r o d gels o f each p e a k s h o w e d multiple b a n d s , w h e r e a s 1 0 % - P A G E S D S r o d gels s h o w e d 3 clear b a n d s . C o m p a r i s o n o f the S D S gels with a G-75 s a m p l e revealed s o m e
\
\
\
c o m m o n b a n d s , t w o over a m o l e c u l a r weight range o f 43,000 a n d 68,000 a n d o n e e s t i m a t e d b e t w e e n 30,000 a n d 35,000. P e a k s I a n d II were t h e n t r e a t e d s e p a r a t e l y in s u b s e q u e n t purification steps; p e a k s III a n d IV were c o m b i n e d a n d f u r t h e r purified. T h e c h r o m a t o f o c u s i n g d a t a in the purification table refers to p e a k s I a n d II since the a m o u n t o f material p r e s e n t in p e a k s III a n d IV was small. Affinity c h r o m a t o g r a p h y using S - h e x y l g l u t a t h i o n e as the i m m o b i l i z e d ligand fulfilled t w o f u n c t i o n s in
\
2-
\
\ \ I
\
\
\
IE u
o
\
"1" ~X
ea
A
-
~ . .
2
4.......___
4
6
8
10
.
12
.
.
.
14
16
Time (hr)
Fig. 1. Chromatofocussing of yeast glyoxalase I. The elution profile shows the fractionation of pooled G-25 material applied to a column (1.0 x 25 cm) containing PBE 96 gel, equilibrated with Tris42H3COOH buffer (25 mM, pH 8.4). Protein was eluted using polybuffer 96 (1:13 dilution factor) adjusted to the limit pH 6.00 with CH3COOH, at a linear flow rate of 12 ml hr -~. - - - , A280;- - . - - , glyoxalase I activity; . . . . . , pH profile.
552
KENNETH T. DOUGLAS et al.
+
Table 2. Carbohydrate composition of yeast glyoxalase I Weight %
I
II
III
IV
Fucose Mannose Galactose Glucose Mannosamine Galactosamine Glucosamine N-Acetyl-neuraminic acid
0.15 0.17 0.16 0.03 0.06 0.17 -0.01 Total
0.75
molecule behaves ideally following a continuous flow pattern and that the shape is of a spherical nature.
I
II
III
IV
Fig. 2. Upper: DISC-PAGE of chromatofocussing peaks I, II, III and IV on 10% polyacrylamide rod gels. Lower: SDS-PAGE of peaks I, II, III and IV on 10% polyacrylamide rod gels. Molecular weights of components present in these peaks were estimated by comparison of standard molecular weight proteins where: 1, bovine serum albumin, 67,000; 2, ovalbumin, 43,000; 3, myoglobin, 17,200; 4, cytochrome c, 11,500. the next step: (I) removal of polybuffer components from the previous step and (2) efficient removal of contaminating proteins utilizing the enzyme's binding activity. The preparation after G-100 SF gel filtration was homogeneous when judged as a single band stained with Coomassie Blue on 15% homogeneous PAGE and 10-18% gradient SDS-PAGE under both normal and overloading sample concentrations. Anodic DISC PAGE on 10% acrylamide slab gels showed a single component, but on rod gels showed two, tight bands. However, on incubation with 100mM GSH (pH 7.0) only one band was detected. Isoelectric points were determined by means of thin layer polyacrylamide plates using standard protein pl markers. The glyoxalase I sample showed 4 major bands at pI's: 6.60, 7.10, 7.25 and 7.75 with two faint bands at 6.80 and 7.65. The number of bands was not reduced by prior incubation with GSH (100mM, pH7.0), KCN (10mM), DTT (50mM). G-100 purified samples of peaks I and II obtained from the chromatography step were identical by this criterion. (b) Ultracentrt[uge data The data obtained for the estimation of S (sedimentation velocity) showed a single peak in the Schlieren pattern and the plot of In r vs time was linear. Data from molecular weight estimation by the sedimentation equilibrium Meniscus depletion method of Yphantis (1964) yielded a linear plot of In C vs (x) 2. Using the appropriate formulae (Bowen and Rowe, 1970), the diffusion coefficient calculated for glyoxalase I was 9.534 x 10 7cm2/sec, with a calculated frictional ratio J'~['o= 1.03, indicating that the
(c) Amino-acid and carbohydrate composition The amino-acid composition of yeast glyoxalase 1 (24, 48 and 72hr hydrolyses) was close to that reported by Marmst~.l and Mannervik, 1978, the main differences being in the Asp, Thr, Ser and Phe contents. In agreement with these authors we also observed routinely 30-33 tryptic peptides on 2-dimensional mapping, in line with a monomeric structure. The total carbohydrate content based on a molecular weight of 3.4 x 104 was 0.75% with fucose, mannose, galactose and galactosamine accounting for 0.65 wt% (see Table 2). (d) Molecular weight of glyoxalase I from yeast The molecular weight of yeast glyoxalase I, determined by SDS-PAGE, was 37,000 from homogeneous 15% PAGE and 35,500 from gradient !(~18% PAGE. The molecular weight was also estimated as 33,800 + 3,300 by gel permeation chromatography on Sephadex G-100 SF (average of 5 samples). In sedimentation velocity experiments a sedimentation coefficient of 3.74+0.115 was obtained. A value of 3.60 ($20,,,.) for the yeast enzyme (Aronsson and Mannervik, 1977) has been reported. The weightaverage molecular weight of yeast glyoxalase I estimated by the Meniscus depletion method of equilibrium ultracentrifugation was found to be 35,800 ___220.
(e) Kinetic parameters With the methylglyoxal/glutathione hemithioacetal as substrate, the following values were obtained at pH6.60, 25°C: kc,t=3.54x 104min i. K,,=5.12_+ 0.33× 10 4M, k , ~ / K , , = 6 . 9 x 107M ~min-'. The values of these parameters are similar to those in other reports in the literature (Aronsson and Mannervik, 1977; Van der Jagt and Hart, 1973). The magnitude ofkcat/K m (2.30 × 107 M -l sec ') above is based on a K,, value calculated from the total concentration hemimercaptal present. As glyoxalase I has been shown under some conditions to use only one of the diastereomeric forms of this hemimercaptal, the actual value of k~,t/K,,, may be greater (~4.6 x 107 M -I sec t). In either case glyoxalase I is one of the most catalytically active of enzymes with a k ~ S K m value close to that set by the diffusion-limit (Fersht, 1977).
Yeast glyoxalase I
(A)
553 e
(B)
I
A I
52
4
%2
7
% b.q
<] 2 2 I
L
n
J
I
200
I
250
250 Wavelength
Wavelength
(nm)
300 (nm)
A
'E u (Cl
7 1.o o c:: ',v 0.8 ,,~ 0.6 I ~ 0.4 4~
0.2 £a
?,
\.
•
I
I
I
250 Wavelength
:k ,
- ~qro.4~-...I 300
(nm)
Fig. 3. (A) Near- and (B) far-UV CD spectra of yeast glyoxalase l (0.5 mg/ml 0.05 M KH2PO4 buffer, pH6.80 and 0.08% v/v, dmso as co-solvent at 23.5°C) in the presence and absence of S-(4bromobenzyl)glutathione (5.2 x l0 5 M, I). Near-UV and far-UV spectra were recorded in duplicate in 0.2 and l0 mm pathlength cells, respectively. There was no significant spectral shift in the far-UV region in the presence of inhibitor. (C) Aromatic CD difference spectrum of yeast glyoxalase I [+ and S-(4-bromobenzyl)glutathione]. -
(f) Circular dichroic (CD ) spectra of yeast glyoxalase I The CD spectra shown in Fig. 3 were recorded in the presence and absence of the strong competitive inhibitor, S-(4-bromobenzyl)-glutathione. The binding of the competitive inhibitor has no major effect on the global secondary structure (considering the region below 220 nm). For the native protein no one conformation (~, fl or random coil) dominates. The (240-300 nm) CD spectrum of yeast glyoxalase I exhibits bands at 264, 281 and 293 nm which are almost certainly attributable to phenylalanine, tryptophan and tyrosine. CD band positions and intensities in this region were found to be temperature independent over the range 23.5-43°C. However, binding of S-(4-bromobenzyl)-glutathione caused small changes in C D intensities with a slight red shift in the 264 nm band and a blue shift in the 293 nm band. The CD difference spectrum (Fig. 3C) has given rise to a positive maximum at 275 rim. The overall shape is similar to CD spectra of tyrosine model compounds (Strickland et al., 1970). The difference spectrum reflects perturbations coincident with the binding of competitive inhibitor. The location of the 275 nm CD maximum leads us to tentatively interpret it as reflecting a tyrosine perturbation, but insufficient fine structure is present to
allow unambiguous assignment. The spectrum possesses no maxima >286 nm, therefore, tryptophan perturbations, if present, are not identifiable. (g) Dependence of K i values The inhibition of yeast glyoxalase I by S-(pbromobenzyl)glutathione has been established to be competitive (Marmst~l and Mannervik, 1979; Douglas et al., 1982) as it has for S-(pazidophenacyl)glutathione (Seddon and Douglas, 1980). For the former Ki was essentially pH independent in the range 5.80-8.00 (Douglas and Ghobt-Sharif, in preparation). For S-(pbromobenzyl)glutathione the mean Ki value of the pH range 5.80-8.00 was found to be 3.41 _ 0.80/aM. For S(p-azidophenacyl)glutathione Ki values were as follow (pH in parentheses): 0 . 7 0 + 0 . 0 3 x 10-4M (5.50), 1.05 _ 0.05 x 10 -4 M (6.60), 1.08 _ 0.08 x 10-4M (7.52): mean value 0.94+__0.21 × 10-4M. DISCUSSION
The purification procedure described gives an apparently homogeneous enzyme from yeast, as judged by SDS, DISC PAGE, chromatographic, ultracentrifugal analysis criteria. The enzyme was purified about 600-fold in a yield of 6.3% to a specific activity
554
KENNETH T. DOUGLASet al.
of 1042 units/mg of protein and was stable when stored at - 20°C. The specific activity achieved agrees well with literature values (Davis and Williams, 1966; Van der Jagt and Han, 1973; Marmst~l and Mannervik, 1978). The molecular weight of the enzyme, by gel permeation chromatography and equilibrium centrifuge analysis, was found to be 33,800 + 3,300 and 35,800_+ 220, respectively. These values are in good internal agreement and with literature estimates (from gel filtration and/or sedimentation velocity estimates) of 32-35,000 (Van der Jagt and Han, 1973; Mannervik et al., 1972; Marmst~l and Mannervik, 1978). The presently-determined sedimentation coefficient (3.74 s) and the more or less spherical shape of the protein molecule, indicated by the close to unity value of the frictional ratio ( f i f o = 1.03) is in agreement with the results of another study (Aronsson and Mannervik, 1977). The isoelectric point (pI) of yeast glyoxalase I has been estimated (Marmsffd et al., 1979) as ~ 7 from an isoelectric focusing column (eluted fractions were assayed for glyoxalase I activity which came out in 43 ml of a 110 ml total column eluant; pH gradient 8 5). Clearly, the resolution/precision of such a procedure is low. Analytical electrofocusing on gels of the present preparation gave 4 major protein bands (pI's 6.60, 7.10, 7.25 and 7.75). Several isoelectrophoretic forms have been reported for glyoxalase I isolated from mammalian sources (Marmst~.l et al., 1979; Uotila and Koivusalo, 1975, 1980). This apparent heterogeneity was lifted by prior treatment with GSH in many cases, but this had no effect on the electrophoretic results given by fresh haemolysates (Uotila and Koivusalo, 1980) and this yeast preparation. These multiple bands may represent true heterogeneity or may be artefactual. Aronsson and Mannervik (1977) have suggested these isoelectrophoretic forms may originate from a mixed disulphide of the enzyme and glutathione. However, while the yeast enzyme has accessible SH groups, the mammalian enzymes (also with multiple bands on electrofocussing) do not (Ekwall and Mannervik, 1970; Mannervik et al., 1975). Glycoproteins may exhibit multiple bands if the sugar residues present are inhomogeneous. In the present study yeast glyoxalase I on carbohydrate analysis revealed a small, but significant carbohydrate content (0.75% by weight) representing an average of 2 sugar units per enzyme molecule. Mannose and galactosamine are typically found associated with yeast proteins, linked via an O-glycosidic linkage through the hydroxyl group of serine or threonine (Gottschalk, 1972; Sharon, 1975). The presence of a carbohydrate component in yeast glyoxalase I has not previously been reported. The multiple bands on electrofocussing of yeast glyoxalase I may arise in part from this polydispersity caused by variable sugar contents of individual molecules. The carbohydrate analysis (Table 2) reveals 7 difJerent types of sugar (the small amount of glucose present may be derived from the final Sephadex G-100 step). As there are on average only 2 sugar residues per protein molecule, the preparation must by polydisperse in this respect of sugar content. Clearly from the data in this work [for S-(p-azidophenacyl)glutathione and S-(p-bromoben-
zyl)glutathione] as well as that reported for S-(mtrifluoromethylbenzyl)glutathione and N-acetyI-S(p-bromobenzyl)glutathione (Douglas et al., 1982a) there is little or no effect of pH on the K, values for competitive inhibitors possessing the glutathione backbone structure. This stands in marked contrast to the pH dependence of Km reported for glutathione hemimercaptals formed with methylglyoxal, phenylglyoxal and p-chlorophenylglyoxal (Van der Jagt and Han, 1973). The magnitude of Km depends on two ionisations of pKapv values 4.7-5.2 and 8.4-8.9, depending on the substrate. For all three substrates Vmax was pH-insensitive, which was suggested to indicate that in this pH range no ionisable groups occur which are important in the conversion of substrate to product. The groups observed for K,, were ascribed to enzymatic ionisations rather than dissociations associated with glutathione, with the group at pKapp ~ 5 ascribed to a ~ O 2 H site and that at ~ 8.7 to an - - N H + group (Vander Jagt and Han, 1973). An apparent related anomaly for human erythrocyte glyoxalase I is that the K,, value for the adduct of methylglyoxal and glutathione was effectively unchanged by substitution of Co 2+, Mn 2+ or Mg 2+ for the crucial Zn z+ of the native enzyme, whilst the competitive inhibition constant (K~) for S-(pbromobenzyl)glutathione was sensitive to the nature of the metal ion (Aronsson et al., 1981). Consider the simple scheme for enzyme catalysis of equation (2) for which K m = ( k 2 + k 3 ) / k ~ and K s = k2/k I (the enzyme-substrate complex dissociation constant). kl
k3
E + S~ES--* E + P
(2)
k2
For competitive inhibition, we also have equation (3) with K~ = ks~k4. k4
E + I~-~--EI k5
(3)
If S and I bind to the same site then clearly K,, --~ K~ for this enzyme as both I and S would have similar pH dependencies, which is not observed. Nor is this model possible with K,, = (k: + k3)/k~ with the pH dependence of Km ascribed to the k 3 term (not present in Ki) as Vm,x(=k3[E0]) has no pH-dependence. If, as Vander Jagt and Han (1973) argue, K,, = k3/k ~, a problem arises in the pH dependence unless k~ and k2 have equal but opposite pH dependences which cancel (as k4 and k 5 pH dependencies must have balanced each other for K0. The dispersion in metal ion sensitivity of Km and K, may arise if the metal ion does not lie in the activesite, but there are good circumstantial arguments against this (Aronsson et al., 1981). Alternatively Ki may be wrongly defined for glutathione analogues. The equilibrium binding constant of S-(pbromobenzyl)glutathione is not significantly different from its steady-state inhibition constant (l/K,) (Marmst~.i and Mannervik, 1979). However, although glutathione does not affect the equilibrium binding of S-(p-bromobenzyl)glutathione it competes directly with this S-blocked analogue under turnover conditions. This was explained by Marmsthl and Mannervik (1979) by suggesting that glyoxalase I exists in an "equilibrium" form which does not bind
Yeast glyoxalase I glutathione and a "catalytic" conformation which does. The C D spectrum of glyoxalase I is slightly sensitive to S-(p-bromobenzyl)glutathione in the aromatic region and it is possible from the results that binding of this inhibitor perturbs the environment of a tyrosine residue. This tends to support the view of Marmsffd and Mannervik with the different enzyme conformations being inhibitor inducible although a minor configurational change is all that is necessary to explain the C D results. SUMMARY
Glyoxalase I (EC 4.4.1.5) has been purified ~600-fold from baker's yeast by a procedure involving chromatofocussing and affinity chromatography. The enzyme contained 0.75% w/w carbohydrate consisting of fucose, mannose, galactose, mannosamine, galactosamine, N-acetylneuraminic acid and possibly glucose. The molecular weight was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis to be ~36,000, 33,800 + 3300 by gel filtration and 3 5 , 8 0 0 _ 200 by equilibrium centrifugation. The sedimentation coefficient was 3.74 s. F o u r isoelectric forms were detected with pI values of 6.60, 7.10, 7.25 and 7.75, probably because of the polydispersity arising from the sugar component. The circular dichroic spectra were measured in the absence and presence of the competitive inhibitor S-(p-bromobenzyl)glutathione, the presence of which appeared to perturb a tyrosine residue. The effect of pH on Ki for this inhibitor was measured and pH effects on glyoxalase I discussed. Acknowledgements--We are grateful to the Medical Re-
search Council for support (A.P.S.) and to Drs R. C. Hider and A. Drake for assistance in the CD aspects. REFERENCES
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