Negative co-operativity in glutamate dehydrogenase - Europe PMC

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Biochem. J. (1985) 225, 209-217 Printed in Great Britain

Negative co-operativity in glutamate dehydrogenase Involvement of the 2-position in glutamate in the induction of conformational changes

Evelyn T. BELL, Charles LiMUTI, Cheryl L. RENZ and J. Ellis BELL Department of Biochemistry, University of Rochester Medical Center, Box 607, 601 Elmwood Avenue, Rochester, NY 14642, U.S.A.

(Received 16 July 1984/Accepted 7 September 1984) The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)Hsubstrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.

Initial-rate studies of the oxidative deamination of glutamate by bovine liver glutamate dehydrogenase (EC 1.4.1.3) with either NAD+ or NADP+ as the varied substrate gave plots that were nonlinear (Dalziel & Engel, 1968; Engel & Dalziel, 1969), and it was proposed that negative cooperativity among the six apparently identical polypeptide chains of the enzyme could result in such behaviour. Subsequent re-analysis of the data of Dalziel and Engel (Engel & Ferdinand, 1973) suggested that the interactions must include positive and negative components, and that they must involve catalytic steps as well as coenzymebinding steps. Studies of the binding both of oxidized (Dalziel & Egan, 1972) and reduced coenzymes (Melzi D'Eril & Dalziel, 1973; George & Bell, 1980) to the enzyme, on binding to half the subunits in the hexamer, was shown, in the presence of glutarate (an analogue of glutamate), to induce a conformational change in the other half Vol. 225

of the oligomer (Bell & Dalziel, 1973). Although these binding studies, and studies of induced conformational changes, involved the use of glutarate and did not represent the catalytically active enzyme, several further studies have shown that subunit-subunit interactions take place while the enzyme is catalytically functioning (Alex & Bell, 1980; Smith & Bell, 1982). In the original studies by Dalziel & Engel (1968) and in our own subsequent studies (LiMuti & Bell, 1983) it was found that the active monocarboxylic amino acid norvaline was a substrate for the enzyme (although a poor one), but did not exhibit negative cooperativity, and it was suggested that both carboxy groups on glutamate were required to allow the subunit-subunit interactions to take place. It has long been known that two carboxy groups (or equivalent charge densities) appropriately positioned are required for effective amino acid/oxo acid substrate or inhibitor binding (Caughey et al.,

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E. T. Bell, C. LiMuti, C. L. Renz and J. E. Bell

1957; Rogers, 1971; Rogers et al., 1972). Binding studies by Dalziel & Egan (1972) showed that glutarate considerably enhanced binding of oxidized coenzyme to the enzyme, and our own subsequent studies (C. LiMuti & J. E. Bell, unpublished work) have shown that the monocarboxylic equivalent 2-oxovalerate does not. There has been much controversy concerning the potential role of a second, non-active, coenzyme-binding site per subunit. Since the early suggestion (Frieden, 1959) that such a second site existed but did not bind NADP(H), much has been discussed concerning differences between the behaviour with NAD(H) and NADP(H). The studies by Dalziel & Engel (1968), which led to the proposal of negative co-operativity in this enzyme, showed virtually identical behaviour with NAD+ and NADP+, suggesting that the subunit-subunit interactions did not involve this second site. However, despite an accumulation of evidence from a variety of laboratories (see Dalziel, 1975, for a review) that the second coenzyme-binding site does not bind NADPH with anything like the affinity for NADH, and studies showing that binding of NADP+ to a second site is much less effective than for NAD+ (Smith & Bell, 1982; C. LiMuti & J. E. Bell, unpublished work), it is still speculated that a second, oxidized-coenzymebinding, site is involved in the generation of the non-linear Lineweaver-Burk plots that Dalziel & Engel (1968) attributed to negative co-operativity. In the present paper we report kinetic studies, ligand-binding studies and fluorescence-spectral studies which show that the 2-position substituent on L-glutamate or 2-oxoglutarate (substrates for the enzyme) or L-2-hydroxyglutarate is vital for the induction of subunit-subunit interactions that affect catalysis. With glutarate (2-position CH2) only coenzyme binding appears to be affected. Furthermore, identical results are obtained with NAD+ and NADP+ as coenzyme. These experiments provide further evidence that the non-linear Lineweaver-Burk plots obtained with oxidized coenzyme as the varied substrate are, in fact, due to negative co-operativity between the subunits of the enzyme and that this co-operativity involves both a ligand-binding and a catalytic effect.

metrically by using an absorption coefficient at 280 nm of 0.93 cm-1 for a 1 mg/ml solution (Egan & Dalziel, 1971). Concentrations of enzyme used in these experiments are given in the Figure legends, calculated by using a subunit M, of 55700. In a number of cases experiments were conducted at several different enzyme concentrations in the range of 20-220 nM. Identical results were obtained over this concentration range. All substrates, coenzymes, coenzyme analogues, substrate analogues and buffer salts were obtained from Sigma Chemical Co. Solutions were made up with 18 MQ distilled deionized water from a 4 Bowl Milli Q System. Rate measurements were made either by fluorimetry for the oxidative deamination by using excitation at 340nm and measurements of emission at 450nm, or by absorbance measurements at 340nm for the reductive amination reactions by using a millimolar absorption coefficient of 6.22mM- Icm-l (Stein et al., 1963). Fluorescence rate measurements were calibrated from a titration with NADH or NADPH. Initialrate measurements were performed in at least triplicate. Results shown are the averages of the experimental values obtained. Reproducibility of the individual data points was within +6%. Standard deviations for inhibition constants shown in Tables 1 and 2 were estimated by using linear regression of the appropriate LineweaverBurk plots. The following statistical criteria were used in determining the inhibition patterns of the various inhibitors used in this study. If the slopes in the presence and in the absence of the inhibitor differed by more than 2S.D. from each slope the inhibition was considered to be competitive. If the intercepts differed by more than 2S.D. from each intercept, the inhibition was considered to be uncompetitive. If both criteria were met, the inhibition was considered to be non-competitive, and K, values from intercept changes and from slope changes were calculated. Fluorescence titrations and calculation of binding data were performed as described previously (Bell, 1981). Fluorescence spectra are uncorrected for phototube-response variations with wavelength. Emission spectra are shown as molecular emission spectra for the appropriate complex, where contributions from unbound fluorophore to the fluorescence at any given wavelength are subtracted. Data were handled and analysed by using programs written by one of us (E. T. B.) for a HewlettPackard HP87XM personal computer.

Materials and methods Bovine liver glutamate dehydrogenase used in these studies was obtained either as a glycerol solution or as an aq. (NH4)2SO4 suspension from Sigma Chemical Co. Either form was thoroughly dialysed against 0.1 M-sodium phosphate buffer, pH7.0, containing lOuM-EDTA before use. Both forms of the enzyme gave identical results. Enzyme concentrations were measured spectrophoto-

Results and discussion The studies reported in this paper were carried out to allow a comparison of the ability of various analogues of glutamate to bind to glutamate

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Negative co-operativity in glutamate dehydrogenase

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Fig. 1. Inhibition studies, with glutarate, 2-hydroxyglutarate and 2-oxoglutarate, of the oxidative deamination reaction catalysed by glutamate dehydrogenase, with (a) Lglutamate as the varied substrate, and (b) NAD+ as the varied substrate A, No inhibitor; EO, + 5 mM-glutarate; E, + 5 mML-2-hydroxyglutarate; A, + 2mM-2-oxoglutarate. Other conditions: 0.1 M-phosphate buffer, pH 7.0, containing 1QOM-EDTA, at 25°C, with fixed concentrations of L-glutamate (50mM) or NAD+ (150 gM) as appropriate. Glutamate dehydrogenase concentrations between 21 nM and 62 nm were used:

dehydrogenase. As a result of these studies, however, we have obtained further evidence for catalytic-site co-operativity in this enzyme, and new insights into the structural requirements of the substrate that allow this co-operativity to take place. Kinetic studies A variety of dicarboxylic acid analogues of Lglutamate have been used in inhibition studies of the oxidative deamination reaction. These include glutarate, 2-oxoglutarate, L-2-hydroxyglutarate, D2-hydroxyglutarate and D-glutamate. From the results shown in Fig. 1 and Table 1, it is evident that, although glutarate, 2-hydroxyglutarate (L- or D-), D-glutamate or 2-oxoglutarate are all effective inhibitors of the enzyme, there are some differences between the inhibition patterns Vol. 225

211 observed. Whereas glutarate and 2-hydroxyglutarate (either L- or D-) are both strict competitive inhibitors versus glutamate at several NAD+ concentrations, 2-oxoglutarate as a product inhibitor and D-glutamate appear to be non-competitive. Significantly, as shown in Fig. 2, and Table 1, the D-isomers of glutamate and 2-hydroxyglutarate are inhibitors, but significantly less potent than glutarate or L-2-hydroxyglutarate. Similarly, in the reductive amination reaction (Fig. 3 and Table 2) L-glutamate and glutarate are both competitive inhibitors with respect to 2-oxoglutarate, and L-2hydroxyglutarate is a non-competitive inhibitor. From plots with coenzyme concentration varied, glutarate, 2-oxoglutarate and L-2-hydroxyglutarate all appear to be uncompetitive inhibitors of the oxidative deamination reaction at high concentrations of glutamate. Glutarate, L-glutamate and L-2hydroxyglutarate are all non-competitive inhibitors versus NADH. The only remaining difference observed among glutarate, L-glutamate and L-2hydroxyglutarate is in the reductive amination reaction with ammonia as the varied substrate, where an uncompetitive inhibition pattern is observed with glutarate compared with noncompetitive inhibition for the other two compounds. One of the purposes of the study reported here was to compare glutarate and L-2-hydroxyglutarate with the product inhibitors, L-glutamate and 2-oxoglutarate. The inhibition patterns and K1 values for glutamate and 2-oxoglutarate agree well with those previously reported (Engel & Chen, 1975). From the KI values in the oxidative deamination reaction, it is apparent that there is synergistic binding of coenzyme and each of the inhibitors. Over the substrate concentrations used in these experiments the inhibition patterns are consistent with a rapid-equilibrium random-order addition of substrates in the reductive amination reaction, and suggest that there may be a preferred order of addition, of coenzyme followed by glutamate in the oxidative deamination reaction, as has been suggested previously (Rife & Cleland,

1980). The non-linear nature of Lineweaver-Burk plots with varied NAD+ concentrations at high glutamate concentrations was extensively studied by Dalziel & Engel (1968), who attributed the breaks to negative homotropic interactions between the subunits of the enzyme. At low glutamate concentrations (0.25mM) these non-linearities were not observed, leading to the postulate that the interactions occurred in the ternary enzyme-NAD+glutamate complex. We have examined the ability of glutarate, 2-oxoglutarate and 2-hydroxyglutarate (D- or L-) and D-glutamate to act as inhibitors versus NAD+ with low fixed glutamate concentrations. As shown in Fig. 4, the presence of

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Table 1. Inhibition constants for the oxidative deamination reaction catalysed by glutamate dehydrogenase Key to pattems: C, competitive; UC, uncompetitive; NC, non-competitive. KlaPp values are given as means + S.D.; S and I indicate calculated from slope and intercept effects respectively. KfVPVaried Concn. of Inhibitor substrate Pattern fixed substrate (mM) Glutarate Glutamate 150pM-NAD+ 0.76+0.02 C 254uM-NAD+ 3.07 +0.09 C NAD+ 50mM-Glutamate 9.80+ 1.00 UC 2-Oxoglutarate Glutamate 150M-NAD+ NC 0.31 + 0.015 S 1.30+0.18 I 25 uM-NAD+ NC 1.24 + 0.04 S 2.16+0.35 I NAD+ 50mM-Glutamate 1.33+0.16 UC L-2-Hydroxyglutarate Glutamate 1.04+0.06 C 150pM-NAD+ 25 uM-NAD+ 5.50+0.29 C NAD+ 50mM-Glutamate UC 1.50+0.20 5 mM-Glutamate 6.40+0.36 UC D-2-Hydroxyglutarate Glutamate 150pM-NAD+ 4.03+0.14 C NH4C1 Glutamate 150pM-NAD+ 1.80+0.06 C D-Glutamate Glutamate NC 4.20 + 0.17 S 150pM-NAD+ 6.20+0.42 I

0.4

0.6

0.8

1/[Glutamate] (mM-') Fig. 2. Inhibition studies of glutamate dehydrogenase with D- and L-isomers of 2-hydroxyglutarate and D-glutamate A fixed concentration of NAD+ (150pM) was used with L-glutamate as the varied substrate, in the absence of inhibitors (A), or in the presence of 5 mM-L-2-hydroxyglutarate (O), D-2-hydroxy-

glutarate (A) or D-glutamate (U). The glutamate dehydrogenase concentration was 108 nm.

2-oxoglutarate leads to potent inhibition, but also a non-linear plot, shown here as a Lineweaver-Burk plot, similar to those observed with coenzyme concentration varied at high glutamate concentrations. The inset in Fig. 4 shows the data, in the presence of 2-oxoglutarate, in the form of an Eadie-Hofstee plot, where the non-linearity is also clearly observable. These results suggest that formation of a non-catalytically active enzyme-NAD+-2-oxoglutarate complex at

generates

some of the active sites within the hexamer of the enzyme induces a conformational change in the remaining catalytic sites that leads to a relative increase in the oxidative deamination of glutamate. When similar experiments are performed with several concentrations of glutarate (Fig. 5), no induced curvature in the plots is detected either in Lineweaver-Burk plots or in Eadie-Hofstee plots (inset), suggesting that the enzyme-NAD+-glutarate complex is unable to induce the conformational change leading to enhanced catalytic activity of the remaining subunits. These results indicate a role of the 2-position substituent in the induction of subunit-subunit interactions affecting the catalytic activity of the enzyme. Experiments with 2-hydroxyglutarate shed further light on the potential involvement of the 2position in such induced conformational changes. With L-2-hydroxyglutarate (Fig. 6) markedly nonlinear Eadie-Hofstee plots are obtained with NAD+ concentration varied at low glutamate concentrations. However, with D-2-hydroxyglutarate as the inhibitor, linear plots are obtained. These results confirm the above suggestion that there is an involvement of the 2-position substituent in induction of co-operativity, and further suggest that this substituent must be in the correct spatial position for these induced conformational changes to take place. In all of the experiments described so far, similar effects to those described with NAD+ as coenzyme are also observed with NADP+ as coenzyme. Since NADP+ does not bind to the second, 'regulatory',

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Negative co-operativity in glutamate dehydrogenase 12

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Fig. 3. Inhibition studies, with glutarate, 2-hydroxyglutarand glutamate, of the reductive amination reaction catalysed by glutamate dehydrogenase, with (a) 2-oxoglutarate as the varied substrate, (b) with NADH as the varied substrate, and (c) with NH3 as the varied substrate A, No inhibitor; E1, + 5 mM-glutarate; *, + 5 mML-2-hydroxyglutarate; A, + 5mM-glutamate. Other conditions: 0.1 M-phosphate buffer, pH 7.0, containing 1O2UM-EDTA, at 250C, with fixed concentrations of 2-oxoglutarate (0.2mM), NADH (50uM) or NH3 (10mM) as appropriate. Glutamate dehydrogenase concentrations between 20nm and 48nM were used. ate

coenzyme-binding site per subunit with an affinity anything approaching that of NAD+, the observation of similar effects with NAD+ and NADP+ suggest that it is unlikely that this second, 'regulatory', coenzyme-binding site is involved in these processes. Vol. 225

213

In addition to the kinetic experiments discussed above, we have sought other ways to examine the interactions between enzyme, coenzyme and glutamate or glutarate to test further the conclusion that the 2-position substituent may be important in the induction of conformational changes in ternary complexes of the enzyme. From the reduced-coenzyme fluorescence-excitation spectra shown in Fig. 7, it is quite apparent not only that the fluorescence of bound NAD(P)H in the enzyme-NAD(P)H-glutamate ternary complex is much enhanced when compared with that of the binary complex or the enzyme-NAD(P)Hglutarate ternary complex when the coenzyme is directly excited, but that the glutamate-containing complex has a very prominent excitation peak centred around 280-285 nm, indicating a readier energy transfer between protein tryptophan or tyrosine residues in the enzyme-NAD(P)H-glutamate complex than in either the binary complex or the ternary complex containing glutarate. This indicates that glutamate and glutarate in the ternary complex lead to the induction of quite different conformations of the protein. It is also apparent from the emission spectra shown in Fig. 8 that glutamate and glutarate have differing effects on the environment of the bound reduced coenzyme. As in the kinetic studies discussed above, similar results are obtained with NADH and NADPH. Finally, we have made use of fluorescence titrations to examine the binding of reduced coenzyme in the presence of glutarate or glutamate. Fluorescence titrations were performed at several different enzyme concentrations, with varied coenzyme (NADH or NADPH) concentrations in the absence of co-ligands, or in the presence of saturating concentrations of L-glutamate or glutarate (identical results were obtained with either 50mM- or lOOmM-co-ligand). Fig. 9(a) shows a titration, in the presence of L-glutamate, of enzyme with NADPH. Shown in Fig. 9(b) is the Scatchard plot calculated from these data by using a fluorescence enhancement (determined experimentally from a plot of fluorescence versus [glutamate dehydrogenase] with 1 tM-NADPH) of 4.0. Also shown in Fig. 9(b) is a Scatchard plot from a similar experiment with the use of 50mMglutarate. The results of these and other experiments are given in Table 3. An examination of this Table shows several points. (1) In all cases the fluorescence enhancements for NADH and NADPH are approximately the same. (2) The fluorescence enhancements in the presence of glutamate are considerably higher than those determined in the presence of glutarate, consistent with the spectral studies discussed above. (3) In the presence of glutamate, two dissociation constants

E. T. Bell, C. LiMuti, C. L. Renz and J. E. Bell

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Table 2. Inhibition constants for the reductive amination reaction catalysed by glutamate dehydrogenase When 2-oxoglutarate was the varied substrate the reaction mixture also contained 25pM-NADH and 10mM-NH4Cl. When NH3 was the varied substrate the reaction mixture also contained 25gM-NADH and 0.2mM-2-oxoglutarate. When NADH was the varied substrate the reaction mixture also contained 0.2mM-2-oxoglutarate and 10mMNH4Cl. Key to patterns: C, competitive; UC, uncompetitive; NC, non-competitive. KfPP. values are given as means+ s.D.; S and I indicate calculated from slope and intercept effects respectively. Pattern Inhibitor Varied substrate KIPP (mM) C 5.4+0.4 2-Oxoglutarate Glutarate 8.0+0.6 NH3 UC NC 18.0+ 1.2 S NADH 13.0+0.8 I C 3.0+ 0.2 Glutamate 2-Oxoglutarate 17.0+ 1.3 S NC NH3 10.0+0.7 I NADH NC 36.0+2.6 S 8.6+0.7 I 1.0+0.1 S NC L-2-Hydroxyglutarate 2-Oxoglutarate 7.7+0.5 I 11.8+ 1.2 S NC NH3 1.6+0.2 I NADH 2.8 + 0.2 S NC 3.9+0.3 I

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1/[NADH+l (,am-') Fig. 4. Effects of2-oxoglutarate on Lineweaver-Burk plots of the reaction catalysed by glutamate dehydrogenase with NAD+ as the varied substrate at 0.25mM-L-glutamate The oxidative deamination reaction was studied in the absence (A) and in the presence (-) of 5mM-2oxoglutarate with NAD+ concentrations ranging from 10pM to 500pM. Other conditions were as indicated in Fig. I legend. The inset shows the data in the presence of 2-oxoglutarate in the form of an Eadie-Hofstee plot. The glutamate dehydrogenase concentration was 127nM.

0

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l/[NAD+lI (um- 1) Fig. 5. EJiects oj'glutarate on Lineweaver-Burk plots of the reaction catalysed by glutamate dehydrogenase with NAD+ as the varied substrate at 0.25mM-glutamate The oxidative deamination reaction was studied in the absence (A) and in the presence of 1.25 mMglutarate (El), 2.5 mM-glutarate (A) or 5mM-glutarate (G). Other conditions were as indicated in Fig. 4 legend. The inset shows data in the presence of glutarate in the form of an Eadie-Hofstee plot. The glutamate dehydrogenase concentration was 124nM.

required to describe the binding of either NADH or NADPH to a total of six sites per hexamer. That the fluorescence experiments do not detect binding of NADH to its second, nonactive, site per subunit indicates that under the conditions of these experiments binding of NADH to this second site, if it occurs, produces no fluor-

are

enhancement. (4) Glutamate enhances binding of reduced coenzyme to the active site quite considerably compared with glutarate. (5) Glutarate, although it may enhance binding, does not appear to generate the curvature in Scatchard plots seen with glutamate.

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Negative co-operativity in glutamate dehydrogenase

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vl/INAD+]I (s-') Fig. 6. Comparison of the effect of L- and D-2-hydroxyglutarate on Eadie-Hofstee plots of the reaction catalysed by glutamate dehydrogenase with NAD+ as the varied substrate at 0.25mM-L-glutamate The oxidative deamination reaction was studied in the presence of 5 mM-L-2-hydroxyglutarate (A) or of 5mM-D-2-hydroxyglutarate (U). Other conditions were as indicated in Fig. 4 legend. The glutamate dehydrogenase concentration was 218 nm.

On the basis of these binding studies it is quite apparent that, although glutarate on forming an abortive complex with enzyme-NAD(P)H may enhance the binding of reduced coenzyme, as has been shown previously to be the case for oxidized coenzyme (Dalziel & Egan, 1972; C. LiMuti & J. E. Bell, unpublished work), it does not have the same effects as glutamate on the binding of reduced coenzyme. In the presence of glutamate, we and others (George & Bell, 1980; Melzi D'Eril & Dalziel, 1973) have shown strong negative cooperativity for binding of reduced coenzyme to the active site. In protection studies and in c.d. studies it has been shown (Chen & Engel, 1974, 1977) that coenzyme, in the presence of glutarate, induced a conformational change in the protein, and that two appropriately spaced carboxy groups are required. This conformational change, which appears to be associated with the synergistic binding of coenzyme and substrate, is separate from the conformational change inferred from the kinetic studies described in the present paper and demonstrated by the fluorescence-spectral studies reported here. The latter conformational change requires the 2-

Vol. 225

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400

Fig. 7. Fluorescence-excitation spectra ofglutamate dehydrogenase-coenzyme complexes Excitation spectra, with emission monitored at 450nm, were recorded for (a) NADH (5.1 uM) (curve 1), enzyme-NADH (18 pM-glutamate dehydrogenase, 1.02puM-NADH) (curve 2), enzyme-NADH-glutarate (18 pM-glutamate dehydrogenase, 1.02 uM-NADH, 50mM-glutarate) (curve 3) and enzyme-NADH-L-glutamate (18 pM-glutamate dehydrogenase, 1.02 pM-NADH, 50mM-L-glutamate) (curve 4), and (b) NADPH (5.8 pM) (curve 1), enzyme-NADPH (18 pM-glutamate dehydrogenase, 1.16pM-NADPH) (curve 2), enzymeNADPH-glutarate (18 pM-glutamate dehydrogenase, 1.16 pM-NADPH, 50mM-glutarate).

position substituent on the substrate (in the Lform) and appears to be associated with the catalytic co-operativity shown by the enzyme. Interestingly, it has been reported (Hornby & Engel, 1983) that 3-methylglutamate, although a very poor substrate, shows similar 'coenzyme activation' to glutamate. Unlike other alternative substrates for glutamate dehydrogenase, 3-methylglutamate contains two carboxy groups and a 2-position substituent, and this observation emphasizes the requirement of both for co-operativity to take place, and is consistent with our suggestion that the 2-position is involved in the induction of a conformation change in the ternary complex that is associated with catalytic-site co-operativity.

E. T. Bell, C. LiMuti, C. L. Renz and J. E. Bell

216 Ju

In summary, on the basis of initial-rate kinetic studies we have demonstrated that the subunit cooperativity exhibited by glutamate dehydrogenase involves a 2-position substituent on the amino acid/oxo acid substrate, in addition to the two carboxy groups (whose primary function is enhancing the affinity of the enzyme for coenzyme). The 2-position on the substrate appears to be intimately involved in inducing the conformational change between subunits that leads to the catalytic co-operativity seen in this enzyme.

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Table 3. Summary ofbinding datafor glutamate dehydrogenase at pH 7.0: effect of co-ligands Apparent Kd values are given as means+S.D. Fluorescence Apparent Conditions enhancement Kd (pM) NADH No co-ligands 2.0 45 + 8 2.0 10+2.1 50mM-Glutarate 50mM-Glutamate 1.0+0.1 3.5 7.0+0.4 NADPH No co-ligands 2.0 40+7.5 50mM-Glutarate 2.0 9.0+ 1.6 50mM-Glutamate 4.0 0.50+0.06 2.0+0.1

20

10

2

o 400

425

450

475

500

Wavelength (nm) Fig. 8. Fluorescence-emission spectra of glutamate

dehydrogenase-coenzyme complexes Emission spectra, with either 280nm excitation (a) or 340 nm excitation (b), were recorded. Other details were as indicated in Fig. 7 legend.

60

50

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INADPHI (uM) B/lEnzyme] Fig. 9. Fluorescence titration experiments with glutamate dehydrogenase with NADPH in the presence of 5OmM-L-glutamate In (a) NADH concentrations were varied in the absence of enzyme (A) or in the presence of 6.6 pM-glutamate dehydrogenase (A) or 12.4 puM-glutamate dehydrogenase (-). (b) shows a Scatchard plot calculated from the data in (a) for NADH binding in the presence of L-glutamate (A) together with similar data obtained in the presence of

glutarate (L).

1985

Negative co-operativity in glutamate dehydrogenase This work was supported in part by the Doctoral Fellowship Program of Kodak, and by U.S. Public Health Service Grant GM-30195.

References Alex, S.A. & Bell, J. E. (1980) Biochem. J. 191, 299-304 Bell, J. E. (1981) in Spectroscopy in Biochemistry (Bell, J. E., ed.), vol. 1, pp. 155-194, CRC Press, Boca Raton Bell, J. E. & Dalziel, D. (1973) Biochim. Biophys. Acta 309, 237-242 Caughey, W. S., Smiley, J. D. & Hellerman, L. (1957) J. Biol. Chem. 224, 591-607 Chen, S.-S. & Engel, P. C. (1974) Biochem. J. 143, 569574 Chen, S.-S. & Engel, P. C. (1977) Biochem. J. 163, 297302 Dalziel, K. (1975) Enzymes 3rd Ed. 11, 1-60 Dalziel, K. & Egan, R. R. (1972) Biochem. J. 126, 975984 Dalziel, K. & Engel, P. C. (1968) FEBS Lett. 1, 349-352 Egan, R. R. & Dalziel, K. (1971) Biochim. Biophys. Acta 250, 47-49

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217 Engel, P. C. & Chen, S.-S. (1975) Biochem. J. 151, 305318 Engel, P. C. & Dalziel, K. (1969) Biochem. J. 115, 621631 Engel, P. C. & Ferdinand, W. (1973) Biochem. J. 131, 97105 Frieden, C. (1959) J. Biol. Chem. 234, 2891-2896 George, A. & Bell, J. E. (1980) Biochemistry 19, 60576061 Hornby, D. P. & Engel, P. C. (1983) Int. J. Biochem. 15, 495-500 LiMuti, C. & Bell, J. E. (1983) Biochem. J. 211, 99-107 Melzi D'Eril, G. & Dalziel, K. (1973) Monogr. Biochem. 26, 33-46 Rife, J. E. & Cleland, W. W. (1980) Biochemistry 19, 2321-2328 Rogers, K. S. (1971) J. Biol. Chem. 246, 2004-2009 Rogers, K. S., Boots, M. R. & Boots, S. G. (1972) Biochim. Biophys. Acta 258, 343-350 Smith, T. J. & Bell, J. E. (1982) Biochemistry 21, 733-737 Stein, A. M., Lee, J. K., Anderson, C. D. & Anderson, B. M. (1963) Biochemistry 2, 1015-1017