Location of Deuterium Oxide Solvent Isotope Effects in the Glutamate Dehydrogenase Reaction*

Stopped flow studies of D,O kinetic solvent isotope effects on the reaction catalyzed by L-glutamate dehydrogenase reveal, in addition to several effects apparently attributable simply to pK, shifts, a a-fold pH-independent effect on the velocity of the steady state oxidative deamination of L-glutamate by enzyme and NADP. Comparable pH-independent D,O kinetic solvent isotope effects are seen both in a transient phase of the reaction in which cu-ketoglutarate is displaced by L-glutamate from an enzyme-NADPH-cu-ketoglutarate (product) complex and in an analogous model reaction in which oc-ketoglutarate is displaced by n-glutamate. These results suggest that ol-ketoglutarate dissociation from an enzyme-NADPH-cu-ketoglutarate complex is rate-limiting in the steady state. Transient state kinetic studies of the reaction catalyzed by bovine dehydrogenase productive dead-end complexes the deamination of

Stopped flow studies of D,O kinetic solvent isotope effects on the reaction catalyzed by L-glutamate dehydrogenase reveal, in addition to several effects apparently attributable simply to pK, shifts, a a-fold pH-independent effect on the velocity of the steady state oxidative deamination of L-glutamate by enzyme and NADP.
Comparable pH-independent D,O kinetic solvent isotope effects are seen both in a transient phase of the reaction in which cu-ketoglutarate is displaced by L-glutamate from an enzyme-NADPH-cu-ketoglutarate (product) complex and in an analogous model reaction in which oc-ketoglutarate is displaced by n-glutamate. These results suggest that ol-ketoglutarate dissociation from an enzyme-NADPH-cu-ketoglutarate complex is rate-limiting in the steady state.
Transient state kinetic studies of the reaction catalyzed by bovine liver L-glutamate dehydrogenase have produced a detailed picture of the enzyme mechanism in terms of both catalytically productive and dead-end complexes of enzyme, coenzyme, and substrate (l-6). The three kinetic phases (7) of the oxidative deamination of L-glutamate by glutamate dehydrogenase and NADP (hereafter called the forward reaction) are shown schematically in Fig. 1, along with a summary of the mechanism as it is currently understood. The complexes associated with each phase are located below that phase in the figure. The point of entry of ammonium ion into the catalytic scheme has not yet been elucidated.
No significant transient features have been detected for the reverse catalytic reaction (the reductive amination of a-ketoglutarate). It has commonly been assumed that the rate-limiting step in Phase 3 ( Fig. 1) of the forward reaction is the release of bound NADPH from the dead-end enzyme-NADPH-L-glutamate complex (3,4,6). There is evidence from isotope exchange experiments, however, that a-ketoglutarate dissociation may be rate-limiting under similar conditions (8). In the present work, we determine the phenomenological location and pH dependence of deuterium oxide (D,O) kinetic solvent isotope effects in the glutamate dehydrogenase-catalyzed reaction. As was true of the original study of this type by Kosicki and Srere on the steady state kinetics of citrate-condensing enzyme (9), we find that some of the D,O kinetic solvent isotope effects observed here are attributable simply to pK, shifts; but in addition, we find a pH-independent effect velocity of the reverse reaction exhibits pH-dependent D,O solvent isotope effects (Fig. 2b). The pH dependence of the observed D,O kinetic solvent isotope effects is independent of enzyme concentration over a broad range (0.008 mg/ml to 1.0 mdml). D,O Solvent Isotope Effects on Rates of Observable Transients- Fig.  2c shows the pH (pD) dependence of the initial velocity of Phase 1 of the forward reaction (5) in H,O and in 90% D,O. The principal effect appears to arise from a pK, shift. ' Phase 2 of the forward reaction may be isolated in experiments having NADP concentrations sufficiently low that essentially no free NADPH is released in solution. The time course of such an experiment monitored at three wavelengths is shown in Fig. 3. The slow drop in absorbance at 320 nm accompanied by the commensurate increase of absorbance at 363 nm clearly illustrates the blue to red spectral shift. The slow transient observed in these experiments is exponential and the approximately 2-fold solvent isotope effect on the apparent first order rate constant is independent of pH within experimental error (Fig. 4a)

DISCUSSION
The observed D,O solvent isotope effects on the initial velocity of the reverse reaction appear to arise principally from pK, shifts with change of solvent composition.
Since pK, increases in D,O of 0.3 to 0.6 unit are quite common for weak acids which ionize in the experimental pH (pD) range (16), it is not yet possible to say where specifically the D,O effects on the reverse reaction originate.
The principal finding of the present study is the probable mechanistic location of the pH-independent D,O solvent isotope effect on the steady state velocity (Phase 3) of the forward reaction. The lack of other than a strongly pH-dependent D,O effect on the initial velocity of Phase 1 rules out the mechanistic steps associated with Phase 1 as the source of the pH-independent effects observed for the steady state velocity (Phase 3). The pH-independent effects of Phase 3, then, must involve steps which follow the hydride transfer step for the forward reaction.
The mechanistic event which immediately follows Phase 1 is the conversion of the blue-shifted enzyme-NADPH-cu-ketoglutarate complex to the red-shifted enzyme-NADPH-L-glutamate dead-end complex (Phase 2). Indeed, this phenomenon shows a 2-fold pH-independent D,O kinetic solvent isotope effect. The model reaction in which n-glutamate is used to displace a-ketoglutarate from its ternary complex with enzyme and NADPH also shows a 2-fold pH-independent D,O kinetic solvent isotope effect. Thus, the D,O kinetic solvent isotope effects for Phase 3, Phase 2, and a-ketoglutarate displacement are identical, suggesting that these processes share a common mechanistic feature. In addition, the pH dependences of the rates for these three processes are quite similar, considering the mechanistic complexity of the observed phenomena, and the isotope effects for all three show a linear dependence on the percentage of D,O.
One step in the enzymatic mechanism which is common to all three phenomena and which could be responsible for the observed behavior is the release of a-ketoglutarate from its ternary complex with enzyme and reduced coenzyme. This step is thought to be rate-limiting in Phase 2 and in the cr-ketoglutarate displacement by n-glutamate and is certainly involved in the mechanism of the steady state production of free NADPH (Phase 3). Thus, the observations above provide direct evidence that cu-ketoglutarate dissociation is a ratelimiting factor in the steady state phase (Phase 3) of the forward reaction. This conclusion gains additional support from the isotope exchange data (8) and is consistent with transient state data obtained in Tris buffer which have led to the suggestion that coenzyme release from the enzyme-NADPH-L-glutamate dead-end complex is actually rate-limiting in the steady state under conditions similar to the ones employed in the present work (6 In conclusion, the following simple mechanistic picture for Phase 3 of the forward reaction is consistent with all the experimental evidence presently available. Under the experimental conditions reported here, the presence of the dead-end enzyme-NADPH-L-glutamate complex renders a fraction of the enzyme active sites catalytically inaccessible. The remainder of the sites catalyze the oxidative deamination of L-glutamate at a velocity limited by the rate of dissociation of cu-ketoglutarate from a tight enzyme-NADPH-n-ketoglutarate complex (17). The linear dependence of the rate of cY-ketoglutarate dissociation on D,O concentration suggests the possibility that only a single proton is transferred during tu-ketoglutarate dissociation (18). It has been pointed out, however, that in a complex system such as this, with a multiplicity of exchangeable hydrogens, an apparently linear dependence can be generated by a combination of secondary isotope effects (19). If we assume that only a single proton is involved, the isotope effect for cy-ketoglutarate dissociation corresponds either to a primary deuterium kinetic isotope effect with a minimum value of 2.7 * 0.3 or to a pK, shift of at least 0.4 for a group with a pK, above 10.
isotope effect and pH dependence of the velocity of the steady state phase of the catalytic reaction.