Structure of isocitrate dehydrogenase with alpha-ketoglutarate at 2.7-A resolution: conformational changes induced by decarboxylation of isocitrate

The structure of the isocitrate dehydrogenase (IDH) complex with bound alpha-ketoglutarate, Ca2+, and NADPH was solved at 2.7-A resolution. The alpha-ketoglutarate binds in the active site at the same position and orientation as isocitrate, with a difference between the two bound molecules of about 0.8 A. The Ca2+ metal is coordinated by alpha-ketoglutarate, three conserved aspartate residues, and a pair of water molecules. The largest motion in the active site relative to the isocitrate enzyme complex is observed for tyrosine 160, which originally forms a hydrogen bond to the labile carboxyl group of isocitrate and moves to form a new hydrogen bond to Asp 307 in the complex with alpha-ketoglutarate. This triggers a number of significant movements among several short loops and adjoining secondary structural elements in the enzyme, most of which participate in dimer stabilization and formation of the active-site cleft. These rearrangements are similar to the ligand-binding-induced movements observed in globins and insulin and serve as a model for an enzymatic mechanism which involves local shifts of secondary structural elements during turnover, rather than large-scale domain closures or loop transitions induced by substrate binding such as those observed in hexokinase or triosephosphate isomerase.     

Transient-state kinetic analysis of the oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase

The oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase has been analyzed by transient-state kinetic methods and kinetic isotope effect measurements. The reaction time courses show a burst of NADH formation prior to the attainment of the steady-state velocity. The binding of the inhibitor tartronate to the enzyme was examined by monitoring the quenching of the protein's intrinsic fluorescence; the tartronate concentration dependence of the observed rate constant for association was hyperbolic, supporting a two-step model for inhibitor binding. Analysis of the time courses for D-malate oxidation yielded values for many of the microscopic rate constants governing the reaction. The range of possible solutions for the microscopic rate constants was constrained by comparison of the time course for oxidation of unlabeled malate with that of deuterated malate; this analysis relied on the determination of the intrinsic isotope effect on hydride transfer via measurement of D(V/K), T(V/K), and the oxaloacetate partition ratio. The results of the transient-state kinetic analyses suggest that the rate of D-malate oxidation is largely limited by the rate of decarboxylation of the intermediate oxaloacetate which occurs at 11 s-1. Hydride transfer from D-malate to NAD+ occurs with a rate constant of 300 s-1, and (D)k for this step is 5.5. The agreement between experimentally measured steady-state kinetic parameters and kinetic isotope effects and their values calculated from the microscopic rate constants derived from the transient-state kinetic analyses was quite good.     

Inhibition of pigeon breast muscle alpha-ketoglutarate dehydrogenase by structural analogs of alpha-ketoglutarate

Inhibition of alpha-ketoglutarate dehydrogenase (KGD) by dicarboxylates with (oxaloacetate and ketomalonate) and without (malonate, succinate, and glutarate) alpha-keto group was studied. Ketodicarboxylates at low concentrations inhibit KGD in competitive manner. Increase in their concentrations results in appearance of the noncompetitive component. The extent of KGD inhibition by keto dicarboxylates increases with structural similarity of the inhibitor and the substrate, irrespective of preliminary incubation of the enzyme with the inhibitor. This is indicative of blocking the substrate-binding site of KGD by dicarboxylates. In contrast, inhibitory effect of dicarboxylates which contain no keto group increases as their structural similarity with the substrate decreases. Saturation of KGD with dicarboxylates of this type does not completely suppress the enzymatic activity. Alternatively, these analogs display competitive mode of inhibition. Analysis of the data obtained suggests that these dicarboxylates produce catalytically active triple complex keto substrate-KGD-dicarboxylate and that KGD which enters the composition of such a complex inhibits a decreased affinity for the keto substrate as a result of the inhibitor binding.     

Selective inactivation of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase: reaction of lipoic acid with 4-hydroxy-2-nonenal

Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting alpha-ketoglutarate dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate dehydrogenase (PDH), while other NADH-linked dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in the presence of substrates that reduce the sulfur atoms of lipoic acid covalently bound to the E2 subunits of KGDH and PDH. In addition, loss of enzyme activity induced by HNE correlated closely with a decrease in the availability of lipoic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indicated that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the presence of substrates. These results therefore identify a potential mechanism whereby free radical production and subsequent lipid peroxidation lead to specific modification of KGDH and PDH and inhibition of NADH-linked mitochondrial respiration.     

Brain alpha-ketoglutarate dehydrogenase complex activity in Alzheimer's disease

We measured the activity of the alpha-ketoglutarate dehydrogenase complex (alpha-KGDHC), a rate-limiting Krebs cycle enzyme, in postmortem brain samples from 38 controls and 30 neuropathologically confirmed Alzheimer's disease (AD) cases, in both the presence and absence of thiamine pyrophosphate (TPP), the enzyme's cofactor. Statistically significant correlations between brain pH and lactate levels and alpha-KGDHC activity in the controls were observed, suggesting an influence of agonal status on the activity of alpha-KGDHC. As compared with the controls, mean alpha-KGDHC activity, with added TPP, was significantly (p < 0.005) reduced in AD brain in frontal (-56%), temporal (-60%), and parietal (-68%) cortices, with the reductions (-25 to -53%) in the occipital cortex, hippocampus, amygdala, and caudate failing to reach statistical significance. In the absence of exogenously administered TPP, mean alpha-KGDHC activity was reduced to a slightly greater extent in all seven AD brain areas (-39 to -83%), with the reductions now reaching statistical significance in the four cerebral cortical areas and hippocampus. A statistically significant negative correlation was observed between alpha-KGDHC activity and neurofibrillary tangle count in AD parietal cortex, the brain area exhibiting the most marked reduction in enzyme activity; this suggests that the enzyme activity reduction in AD brain may be related to the disease process and severity. In each brain area examined, TPP produced a greater stimulatory effect on alpha-KGDHC activity in the AD group (23-280% mean stimulation) as compared with the controls (-4 to +50%); this TPP effect could be explained by reduced endogenous TPP levels in AD brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro oxidative decarboxylation of L-2-hydroxy-4-methylthiobutanoic acid by L-2-hydroxy acid oxidase A from chicken liver

The aim of this study was to assess prospectively acid-base changes after severe birth acidaemia. Fourty-five term babies with severe acidaemia (median umbilical artery pH 6.99 [Range 6.74-7.05], mean base deficit 16.3 [SD 3.7] mmol/l) were prospectively identified. Pathological cardiotocographs were present in 32 (71%) prior to delivery and 39 (87%) were delivered operatively; 27 for fetal distress. Sixteen required intubation. At one hour of age, median pH was 7.29 [Range 7.04-7.45] and the change in pH correlated with one hour pCO2 (r = 0.62 p < 0.001). pH measurements were obtained in 11 of the 16 babies with a 1 hour pH < or = 7.25 and all values had recovered by this time. Five of this group were receiving oxygen. Of the 11 babies admitted to NICU, 1 died and 3 had evidence of encephalopathy, all of which were normal at follow-up [2-12 months]. Recovery of pH after severe birth acidaemia was evident at 1 hour of age and would appear to be complete by 4 hours.     

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by ferrylmyoglobin

This article describes the development and psychometric evaluation of self-report measurement instruments in (nursing) research. The aim of this study is to gain more insight into, and understanding of the use of such instruments. To be more specific, this paper deals with: (1) What is a self-report measurement instrument?; (2) How to develop such an instrument?; (3) What is its psychometric quality in terms of validity and reliability?; (4) How to analyze an instrument statistically?; (5) Which are the pros en cons of a self-report measurement instrument in general?; and (6) Where do we find examples of good measurement instruments? These six questions will be answered with the help of several practical research examples. The article concludes with a few suggestions for literature concerning existing measurement instruments and their psychometric qualities.     

Effect of C-4'-modification of thiamine pyrophosphate on its coenzyme activity in the oxidative decarboxylation of pyruvic acid reaction

Interaction was studied between pyruvate dehydrogenase (EC 1.2.4.1) and C-4'-substituted analogs of thiaminpyrophosphate, 4'-N (CH3)-TPP, 4'-N(CH3)2-TPP and OH-TPP. None of these analogs was found to replace TPP during the reduction of NAD and 2.6-dichlorophenol-indophenol as well as pyruvate decarboxylation. The decarboxylase activity of the pyruvate dehydrogenase component isolated from the pyruvate dehydrogenase complex was determined according to the 14CO2 yield and production of 2-C-oxoethyl-TPP using 1-14C-pyruvate and 2-14C-pyruvate, as substrates, respectively. All the analogs were found to competitively inhibit pyruvate dehydrogenase, Ki values for 4'-N(CH3)-TPP, 4'-N(CH3)2-TPP and 4'-OH-TPP being 4.1 X 10(-5) M, 8.5 X 10(-5) M and 2.9 X 10(-6) M, respectively; Km values for TPP was equal to 1-2 X 10(-7) M. It is assumed that the analogs of the holoenzymic complex formed by the pyruvate dehydrogenase component of the pyruvate dehydrogenase complex with mono-, dimethyl-TPP and oxo-TPP do not bind the substrate.     

Immunochemical characterization of the deficiency of the alpha-ketoglutarate dehydrogenase complex in thiamine-deficient rat brain

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate-dependent, mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD-induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features.     

Ascorbic acid-dependent turnover and reactivation of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase using thiophenoxyacetic acid

The first step in catabolism of the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is catalyzed by 2,4-D/alpha-ketoglutarate (alpha-KG)-dioxygenase (TfdA) in Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134. This oxygen- and ferrous-ion-dependent enzyme couples the oxidative decarboxylation of alpha-KG (yielding CO2 and succinate) with the oxidation of 2,4-D to produce 2,4-dichlorophenol and glyoxylate. TfdA was shown to utilize thiophenoxyacetic acid (TPAA) to produce thiophenol, allowing the development of a continuous spectrophotometric assay for the enzyme using the thiol-reactive reagent 4,4'-dithiodipyridine. In contrast to the reaction with 2,4-D, however, the kinetics of TPAA oxidation were nonlinear and ascorbic acid was found to be required for and consumed during TPAA oxidation. The ascorbic acid was needed to reduce a reversibly oxidized inactive state that was formed by reaction of the ferrous enzyme with oxygen, either in the absence of substrate or in the presence of TPAA. The dependency on this reductant was not due to an uncoupling of alpha-KG decarboxylation from substrate hydroxylation, as has been reported for several other alpha-KG-dependent hydroxylases. Significantly, the rate of formation of this reversibly oxidized species was much lower when the enzyme was turning over 2,4-D. Evidence also was obtained for the generation of an inactive enzyme species that could not be reversed by ascorbate. The latter species, not associated with protein fragmentation, arose from an oxidative reaction that is likely to involve hydroxyl radical reactions. On the basis of initial rate studies, the kcat and Km values for TPAA were estimated to be 20-fold lower and 80-fold higher than the corresponding values for 2,4-D. The results are incorporated into a model of TfdA reactivity involving both catalytic and inactivating events.     

Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.     

Inactivation and conformational changes of yeast alcohol dehydrogenase in trifluoroethanol solutions

Conformational changes of yeast alcohol dehydrogenase in trifluoroethanol solutions have been followed by fluorescence emission and circular dichroism spectroscopy. At low concentration (less than 5%), trifluoroethanol shows a reversible inhibition competitive to ethanol and noncompetitive to NAD+. The inhibition constants for native and structural-zinc-removed yeast alcohol dehydrogenase were 5.8 and 1.1 mM, respectively, suggesting that the active site becomes more flexible after the structural zinc is removed. At higher trifluoroethanol concentrations the enzyme was irreversibly inactivated. Comparison of inactivation and conformational changes of yeast alcohol dehydrogenase denatured in trifluoroethanol solutions shows that the extent of inactivation is larger than the extent of conformational changes at the same trifluoroethanol concentration. The results obtained from circular dichroism spectra show that the presence of trifluoroethanol can induce the formation of secondary structure of the enzyme.     

Structural damage to lactate dehydrogenase during copper iminodiacetic acid metal affinity chromatography

The stability of the enzyme lactate dehydrogenase (LDH) was evaluated by measuring structural damage and activity loss after exposure to copper-iminodiacetic acid (IDA) immobilized metal affinity chromatography (IMAC) under oxidizing conditions at pH 7.0. Oxidizing conditions were produced by adding reductants commonly employed in bioprocessing and biomedical applications (glutathione, beta-mercaptoethanol, dithiothreitol, cysteine, or ascorbate) and/or hydrogen peroxide to the mobile phase. Most of these additives have been shown recently to give rise to metal-catalyzed oxidation (MCO) reactions on copper-iminodicaetic acid IMAC columns. Structural damage in the form of increased susceptibility to proteolytic degradation, fragmentation, and cross-linking were measured. Increased sensitivity to proteolysis was significant in virtually all cases tested, even when activity remained high (>95% specific activity recovered). In contrast fragmentation and cross-linking were minimal in all cases, even when activity was low (<50%). As the damage was believed to have been caused primarily by MCO reactions, preventative measures consistent with this reaction pathway were tested. The most successful measure for all of the conditions studied was addition of the Cu+ chelating agent bicinchoninic acid (BCA) to the mobile phase. Decreased contact time with the column decreased damage in the case where glutathione was added. Removal of dissolved oxygen by nitrogen sparging and use of Tris-acetate buffer in place of phosphate had no measurable effect. The success of BCA addition in reducing structural damage and activity loss strengthens the conclusion that MCO reactions can occur on copper-iminodiacetic acid IMAC columns. However, the addition of BCA and the other protective measures described were not successful in eliminating the increased proteolytic susceptibility observed when LDH in buffer was exposed to the copper-charged column with no oxidizing additives. This suggests that at least one other pathway for damage exists. This damage is difficult to detect as it did not cause statistically significant losses in enzymatic activity, fragmentation, or cross-linking.     

Inactivation of bacterial D-amino acid transaminase by beta-chloro-D-alanine

Purified D-amino acid transaminase from Bacillus sphaericus catalyzes an alpha,beta elimination from the D isomer of beta-chloroalanine to yield equivalent amounts of pyruvate, chloride, and ammonia; the L isomer of chloroalanine is not a substrate for this transaminase. During the beta elimination there is a synchronous loss in enzyme activity; the Kinact for beta-chloroalanine was estimated to be about 10 micrometers. The alpha-aminoacrylate-Schiff base intermediate formed after beta elimination of chloride ion is probably the key intermediate that partitions between one inactivation event for every 1500 turnovers. In the presence of D-alanine and alpha-ketoglutarate, which are good substrates for the transaminase activity of this enzyme, beta-chloroalanine is a potent, competitive inhibitor (K1 = 10 micrometers) with D-alanine and a weak, uncompetitive inhibitor with alpha-ketoglutarate.     

Inactivation of D-3-hydroxybutyrate dehydrogenase by fumaroyl bis(methyl phosphate)

Fumaroyl bis(methyl phosphate) reacts with the NADH-dependent enzyme, D-3-hydroxybutyrate dehydrogenase, leading to irreversible inactivation. The bifunctional reagent cross-links the subunits of the enzyme. The inactivation is subject to saturation and protection by substrate, consistent with the reaction occurring at the active site. The stoichiometry of inactivation indicates two active sites undergo reaction with each equivalent of reagent. These results indicate that the dimeric enzyme has contiguous active sites. The reagent is likely to react with an active site lysine, consistent with previous suggestions.     

Inactivation of short-chain acyl-coenzyme A dehydrogenase from pig liver by 2-pentynoyl-coenzyme A

2-Pentynoyl-CoA is a mechanism-based inactivator of the flavoprotein short-chain acyl-CoA dehydrogenase from pig liver. Inactivation is associated with the formation of an intermediate absorbing at 800 nm and results in the incorporation of 0.86 +/- 0.13 molecules of radiolabeled inhibitor per subunit. A rapid procedure was devised to isolate the labeled peptide. A glutamate residue was identified as the target of 2-pentynoyl-CoA treatment and proved homologous to the proposed catalytic base, GLU376, in the corresponding medium-chain acyl-CoA dehydrogenase sequence. These results are discussed in terms of the lack of conservation of this glutamate residue in the acyl-CoA dehydrogenase enzyme family.     

Amino acid decarboxylation by bacteria of the genus erwinia under aerobic conditions]

The expression of membrane immunoglobulin (mIg) was examined in three cloned MPC11-derived mouse myeloma cell lines. Membrane immunofluorescence studies demonstrated that IgG2b producer cells (P1) had complete IgG molecules, L-chain producer (L1) had only L-chain determinants and nonproducer (NP2) did not have any Ig determinants on the cell surface. An Ig receptor, with characteristics different from B lymphocyte Fc receptor, has been found to be present on secreting cells (P1 or L1), but not on the NP2 cell variant. The data reported in the present paper indicate that the expression of mIg and of the Ig receptor molecule is clearly correlated with the process of secretion. In the light of previous data reported on Ig secretion, a model is proposed which correlates the process of secretion with the expression not only of mIg, but also of the receptor for Ig.     

Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate

Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mM) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Significant conformational changes (as indicated by the changes of the intrinsic fluorescence emission and the ultraviolet difference spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no significant effect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational flexibility than the enzyme molecule as a whole.     

Human hepatic macrovesicular steatosis: a noninvasive study of mitochondrial ketoisocaproic acid decarboxylation

Differentiating between alcoholic and nonalcoholic hepatic steatosis is often a difficult clinical task. However, decreased fatty acid mitochondrial oxidation appears as the main factor for alcoholic steatosis, whereas nonalcoholic steatosis may be due to other causes. We studied mitochondrial function, based on a 13C-ketoisocaproic acid (13C-KIC) breath test, in nine alcoholic and 12 nonalcoholic steatosis patients and 10 healthy volunteers. Our results showed a 42% 13C-KIC decarboxylation decrease in alcoholic steatosis patients, but not in nonalcoholic steatosis patients. This noninvasive breath test appears helpful for the diagnostic work-up of hepatic steatosis.     

Oxidative decarboxylation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase proceeds by a stepwise mechanism with NADP and APADP as oxidants

Primary kinetic deuterium, 13C, and multiple deuterium/13C-isotope effects on V/K6PG have been measured for the Candida utilis (cu) and sheep liver (sl) 6-phosphogluconate dehydrogenases (6PGDH). With NADP as the dinucleotide substrate, the following values of D(V/K6PG), 13(V/K6PG)H, and 13(V/K6PG)D were measured at pH 8 for cu6PGDH (sl6PGDH): 1.57 +/- 0.08 (1.87 +/- 0.10), 1.0209 +/- 0.0005 (1.0059 +/- 0.000 10), 1.0158 +/- 0.0001 (1.0036 +/- 0.0008). With APADP as the dinucleotide substrate, values for the above isotope effects at pH 8 are as follows: 2.98 +/- 0.08 (2.47 +/- 0.06), 1. 0106 +/- 0.0002 (1.0086 +/- 0.000 09), and 0.9934 +/- 0.0003 (0.9950 +/- 0.0003). Results indicate the oxidative decarboxylation of 6PG to the 1,2-enediol of ribulose 5-phosphate proceeds via a stepwise mechanism with hydride transfer preceding decarboxylation in all cases. The inverse 13C-isotope effect observed with APADP and 6PG-3d may reflect a preequlibrium isotope effect on the binding of 6PG preceding hydride transfer. Deuterium-isotope effects on V, V/KNADP, and V/K6PG are identical at all pHs and for both enzymes. The primary deuterium-isotope effect on V/K6PG for both enzymes is constant at pH values below the pK in the pH profile for V/K6PG, and decreases as the pH increases. Data suggest the development of rate limitation by a step or steps other than the hydride-transfer step as the pH is increased.