Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD? as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 ? resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 ?, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >103-fold increases in V?/K(Lys)E(t) and V?/K(α-kg)E(t) (the double mutation gives >10?-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V?/K(Lys). A value of 2.0 was also observed for (D)(V?/K(Lys))(D?O) when the primary deuterium kinetic isotope effect was repeated in D?O, consistent with a rate-limiting hydride transfer step. A viscosity effect of 0.8 was observed on V?/K(Lys), indicating the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V?/K(Lys)E(t) is pH-independent, which is consistent with the loss of an acid-base catalyst and perturbation of the pK(a) of the second catalytic group to a higher pH, likely a result of a change in the overall charge of the active site. The primary deuterium kinetic isotope effect for H96Q, measured in H?O or D?O, is within error equal to 1. A solvent deuterium isotope effect of 2.4 is observed with NADH or NADD as the dinucleotide substrate. Data suggest rate-limiting imine formation, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. The pH-rate profile for V?/K(Lys)E(t) exhibits the pK(a) for K77, perturbed to a value of ～9, which must be unprotonated to accept a proton from the ε-amine of the substrate Lys so that it can act as a nucleophile. Overall, data are consistent with a role for K77 acting as the base that accepts a proton from the ε-amine of the substrate lysine prior to nucleophilic attack on the α-oxo group of α-ketoglutarate, and finally donating a proton to the imine nitrogen as it is reduced to give saccharopine. In addition, data indicate a role for H96 acting as a general acid-base catalyst in the formation of the imine between the ε-amine of lysine and the α-oxo group of α-ketoglutarate.     

Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.     

Acid-base chemical mechanism of homocitrate synthase from Saccharomyces cerevisiae

Homocitrate synthase (acetyl-coenzyme A:2-ketoglutarate C-transferase; E.C. 2.3.3.14) catalyzes the condensation of AcCoA and alpha-ketoglutarate to give homocitrate and CoA. The enzyme was found to be a Zn-containing metalloenzyme using inductively coupled plasma mass spectrometry. Dead-end analogues of alpha-ketoglutarate were used to obtain information on the topography of the alpha-ketoglutarate binding site. The alpha-carboxylate and alpha-oxo groups of alpha-ketoglutarate are required for optimum binding to coordinate to the active site Zn. Optimum positioning of the alpha-carboxylate, alpha-oxo, and gamma-carboxylate of alpha-ketoglutarate is likely mimicked by the location in space of the 2-carboxylate, pyridine nitrogen, and 4 carboxylate of pyridine 2,4-dicarboxylate. The pH dependence of the kinetic parameters was determined to obtain information on the chemical mechanism of homocitrate synthase. The V profile is bell shaped with slopes of 1 and -1, giving pKa values of 6.7 and 8.0, while V/K(AcCoA) exhibits a slope of 2 on the acidic side with an average pKa value of 6.6 and a slope of -2 on basic side of the profile with an average pKa value of 8.2. The V/K(alpha-Kg) pH-rate profile exhibits a single pKa of 6.9 on the acidic side and two on the basic side with an average value of 7.8. The pH dependence of the Ki for glyoxylate, a competitive inhibitor vs alpha-ketoglutarate, gives a pKa of 7.1 for a group, required to be protonated for optimum binding. Data suggest a chemical mechanism for the enzyme in which alpha-ketoglutarate first binds to the active site Zn via its alpha-carboxylate and alpha-oxo groups, followed by acetyl-CoA. A general base then accepts a proton from the methyl of acetyl-CoA, and a general acid protonates the carbonyl of alpha-ketoglutarate in the formation of homocitryl-CoA. The general acid then acts as a base in deprotonating Zn-OH2 in the hydrolysis of homocitryl-CoA to give homocitrate and CoA. A solvent deuterium kinetic isotope effect of 1 is measured for homocitrate synthase, while a small pH-independent primary kinetic deuterium isotope effect (approximately 1.3) is observed using deuterioacetyl-CoA. Data suggest rate-limiting condensation to form the alkoxide of homocitryl-CoA, followed by hydrolysis to give products.     

Mechanism of proton transfer in the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of (D)V and (D(2)O)V for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, (2)H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys(159) changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on V(max) and V/K(androsterone) and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on V(max) is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Br?nsted relationship between the log(V/K(d)(-base)Et) of the external amine (corrected for molecular size effects) and pK(a) is linear for the K159A mutant-catalyzed reaction at pH 10.4 (beta = 0.85 +/- 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr(155) to Lys(159) via 2'-OH of ribose.     

Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. In this work, we report the kinetic characterization of the MtDHFR. This enzyme has a sequential steady-state random kinetic mechanism, probably with a preferred pathway with NADPH binding first. A pK(a) value for an enzymic acid of approximately 7.0 was identified from the pH dependence of V, and the analysis of the primary kinetic isotope effects revealed that the hydride transfer step is at least partly rate-limiting throughout the pH range analyzed. Additionally, solvent and multiple kinetic isotope effects were determined and analyzed, and equilibrium isotope effects were measured on the equilibrium constant. (D(2)O)V and (D(2)O)V/K([4R-4-(2)H]NADH) were slightly inverse at pH 6.0, and inverse values for (D(2)O)V([4R-4-(2)H]NADH) and (D(2)O)V/K([4R-4-(2)H]NADH) suggested that a pre-equilibrium protonation is occurring before the hydride transfer step, indicating a stepwise mechanism for proton and hydride transfer. The same value was obtained for (D)k(H) at pH 5.5 and 7.5, reaffirming the rate-limiting nature of the hydride transfer step. A chemical mechanism is proposed on the basis of the results obtained here.     

Chemical mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae

Homoisocitrate dehydrogenase (HIcDH, 3-carboxy-2-hydroxyadipate dehydrogenase) catalyzes the fourth reaction of the alpha-aminoadipate pathway for lysine biosynthesis, the conversion of homoisocitrate to alpha-ketoadipate using NAD as an oxidizing agent. A chemical mechanism for HIcDH is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. According to the pH-rate profiles, two enzyme groups act as acid-base catalysts in the reaction. A group with a p K a of approximately 6.5-7 acts as a general base accepting a proton as the beta-hydroxy acid is oxidized to the beta-keto acid, and this residue participates in all three of the chemical steps, acting to shuttle a proton between the C2 hydroxyl and itself. The second group acts as a general acid with a p K a of 9.5 and likely catalyzes the tautomerization step by donating a proton to the enol to give the final product. The general acid is observed in only the V pH-rate profile with homoisocitrate as a substrate, but not with isocitrate as a substrate, because the oxidative decarboxylation portion of the isocitrate reaction is limiting overall. With isocitrate as the substrate, the observed primary deuterium and (13)C isotope effects indicate that hydride transfer and decarboxylation steps contribute to rate limitation, and that the decarboxylation step is the more rate-limiting of the two. The multiple-substrate deuterium/ (13)C isotope effects suggest a stepwise mechanism with hydride transfer preceding decarboxylation. With homoisocitrate as the substrate, no primary deuterium isotope effect was observed, and a small (13)C kinetic isotope effect (1.0057) indicates that the decarboxylation step contributes only slightly to rate limitation. Thus, the chemical steps do not contribute significantly to rate limitation with the native substrate. On the basis of data from solvent deuterium kinetic isotope effects, viscosity effects, and multiple-solvent deuterium/ (13)C kinetic isotope effects, the proton transfer step(s) is slow and likely reflects a conformational change prior to catalysis.     

Effects of high pressure on solvent isotope effects of yeast alcohol dehydrogenase

The effect of pressure on the capture of a substrate alcohol by yeast alcohol dehydrogenase is biphasic. Solvent isotope effects accompany both phases and are expressed differently at different pressures. These differences allow the extraction of an inverse intrinsic kinetic solvent isotope effect of 1.1 (i.e., (D(2(O)))V/K = 0.9) accompanying hydride transfer and an inverse equilibrium solvent isotope effect of 2.6 (i.e., (D(2(O)))K(s) = 0.4) accompanying the binding of nucleotide, NAD(+). The value of the kinetic effect is consistent with a reactant-state E-NAD(+)-Zn-OH(2) having a fractionation factor of phi approximately 0.5 for the zinc-bound water in conjunction with a transition-state proton exiting a low-barrier hydrogen bond with a fractionation factor between 0.6 and 0.9. The value of the equilibrium effect is consistent with restrictions of torsional motions of multiple hydrogens of the enzyme protein during the conformational change that accompanies the binding of NAD(+). The absence of significant commitments to catalysis accompanying the kinetic solvent isotope effect means that this portion of the proton transfer occurs in the same reactive step as hydride transfer in a concerted chemical mechanism. The success of this analysis suggests that future measurements of solvent isotope effects as a function of pressure, in the presence of moderate commitments to catalysis, may yield precise estimates of intrinsic solvent isotope effects that are not fully expressed on capture at atmospheric pressure.     

Chemical mechanism of saccharopine dehydrogenase (NAD+, L-lysine-forming) as deduced from initial rate pH studies

The variation of kinetic parameters with pH has been determined so as to gain insight into the chemical mechanism of the saccharopine dehydrogenase (NAD+,L-lysine-forming)-catalyzed reaction. In the direction of reductive condensation of lysine and alpha-ketoglutarate (reverse reaction), the V/K profile for lysine shows a group with a pK of 6.3 must be unprotonated and a group with a pK of 8.0 must be protonated for activity. Similar pK's are obtained in the pKi profile for ornithine, which acts as a linear competitive inhibitor with respect to lysine. Temperature and solvent perturbation studies show that these groups are probably histidines. The V/K profile for alpha-ketoglutarate reveals a single group with pK = 8.4 (probably lysine) that must be protonated. It is proposed that one of the histidines is involved in the binding of the epsilon-amino group of the substrate lysine and the positively charged lysine residue hydrogen bonds to the carbonyl oxygen of alpha-ketoglutarate. In the direction of saccharopine cleavage, the V/K profile for saccharopine shows that two groups with pK values of 6.0 and 7.1, possibly a histidine and lysine, must be unprotonated for its reaction with the enzyme X NAD+ complex. The log V-pH plots for the forward and reverse reactions both show sigmoidal curves. At low pH, the activity is lower for the forward reaction, and is higher for the reverse reaction. The ionization of a single group appears to be responsible for the change in activity. A tentative scheme for the chemical reaction is presented.     

Uridine diphosphate galactose 4-epimerase. pH dependence of the reduction of NAD+ by a substrate analog

Neutron activation analysis of UDP-galactose 4-epimerase from Escherichia coli for 53 metals shows that the enzyme does not contain any of these metals at significant levels. The substrate analog P1-5'-uridine-P2-glucose-6-yl pyrophosphate (UGP), a structural isomer of UDP-glucose with the sugar linked to UDP through the C-6 hydroxyl group, is an inactivator that irreversibly reduces epimerase.NAD+ to epimerase.NADH. The pH dependence of kobs reveals the essential involvement of an acidic group, kinetically measured pKa = 5.48 +/- 0.08, in unprotonated form and two weakly acidic or basic groups, apparent pKa values of 10.03 +/- 0.43, in protonated forms. Measurements of kobs as a function of [UGP] show that it is given by kobs = k[UGP]/(K + [UGP]) at a given pH, where K = 0.19 +/- 0.04 mM throughout the pH range 4.8-10.4. The pH-dependent first order rate constants range from 0.28 to 1.94 s-1, with the maximum value at pH 7.6 and decreasing at acidic and basic pH values. Reaction of [glucose-1-2H]UGP proceeds with kinetic isotope effects of 5.0, 2.1, 2.0, 1.9, and 3.5 at pH values 5.0, 6.2, 7.6, 9.0, and 10.0, respectively. Therefore, hydride transfer becomes rate-limiting at pH extremes but is not limiting at neutral pH, although deuteride transfer is significantly limiting at all pH values. The isotope effects facilitated correction of the kinetic pK values to the thermodynamic values 6.1-6.2 on the acid side and 9.0-9.6 on the alkaline side. We postulate that the group with pK1 = 5.5 (6.1-6.2 corrected) functions as an enzymic general base that abstracts the glucosyl C-1 hydroxyl proton in concert with transfer of the C-1 hydrogen and two electrons to NAD+. The pH dependence on the alkaline side may be related to the uridine nucleotide-dependent conformational transition that is an essential step in the reduction of epimerase.NAD+ to epimerase.NADH by sugars.     

Mechanisms of enzymatic and acid-catalyzed decarboxylations of prephenate

The prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Escherichia coli catalyzes the oxidative decarboxylation of both prephenate and deoxoprephenate, which lacks the keto group in the side chain (V 78% and V/K 18% those of prephenate). Hydride transfer is to the B side of NAD, and the acetylpyridine and pyridinecarboxaldehyde analogues of NAD have V/K values 40 and 9% and V values 107 and 13% those of NAD. Since the 13C isotope effect on the decarboxylation is 1.0103 with deuterated and 1.0033 with unlabeled deoxoprephenate (the deuterium isotope effect on V/K is 2.34), the mechanism is concerted, and if CO2 has no reverse commitment, the intrinsic 13C and deuterium isotope effects are 1.0155 (corresponding to a very early transition state for C-C bond cleavage) and 7.3, and the forward commitment is 3.7. With deoxodihydroprephenate (lacking one double bond in the ring), oxidation occurs without decarboxylation, and one enantiomer has a V/K value 23-fold higher than the other (deuterium isotope effects are 3.6 and 4.1 for fast and slow isomers; V for the fast isomer is 5% and V/K 0.7% those of prephenate). The fully saturated analogue of deoxoprephenate is a very slow substrate (V 0.07% and V/K approximately 10(-5%) those of prephenate). pH profiles show a group with pK = 8.3 that must be protonated for substrate binding and a catalytic group with pK = 6.5 that is a cationic acid (likely histidine). This group facilitates hydride transfer by beginning to accept the proton from the 4-hydroxyl group of prephenate prior to the beginning of C-C cleavage (or fully accepting it in the oxidation of the analogues with only one double bond or none in the ring). In contrast with the enzymatic reaction, the acid-catalyzed decarboxylation of prephenate and deoxoprephenate (t1/2 of 3.7 min at low pH) is a stepwise reaction with a carbonium ion intermediate, since 18O is incorporated into substrate and its epi isomer during reaction in H218O. pH profiles show that the hydroxyl group must be protonated and the carboxyl (pK approximately 4.2) ionized for carbonium ion formation. The carbonium ion formed from prephenate decarboxylates 1.75 times faster than it reacts with water (giving 1.8 times as much prephenate as epi isomer). The observed 13C isotope effect of 1.0082 thus corresponds to an intrinsic isotope effect of 1.023, indicating an early transition state for the decarboxylation step. epi-Prephenate is at least 20 times more stable to acid than prephenate because it exists largely as an internal hemiketal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tartrate dehydrogenase catalyzes the stepwise oxidative decarboxylation of D-malate with both NAD and thio-NAD

Tartrate dehydrogenase catalyzes the divalent metal ion- and NAD-dependent oxidative decarboxylation of D-malate to yield CO(2), pyruvate, and NADH. The enzyme also catalyzes the metal ion-dependent oxidation of (+)-tartrate to yield oxaloglycolate and NADH. pH-rate profiles and isotope effects were measured to probe the mechanism of this unique enzyme. Data suggest a general base mechanism with likely general acid catalysis in the oxidative decarboxylation of D-malate. Of interest, the mechanism of oxidative decarboxylation of D-malate is stepwise with NAD(+) or the more oxidizing thio-NAD(+). The mechanism does not become concerted with the latter as observed for the malic enzyme, which catalyzes the oxidative decarboxylation of L-malate [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103]. It appears the change in mechanism observed with malic enzyme is specific to its transition state structure and not a generalized trait of metal ion- and NAD(P)-dependent beta-hydroxy acid oxidative decarboxylases. The V/K(malate) pH-rate profile decreases at low and high pH and exhibits pK(a) values of about 6.3 and 8.3, while that for V/K(tartrate) (measured from pH 7.5 to pH 9) exhibits a pK(a) of 8.6 on the basic side. A single pK(a) of 6.3 is observed on the acid side of the V(max) pH profile, but the pK(a) seen on the basic side of the V/K pH profiles is not observed in the V(max) pH profiles. Data suggest the requirement for a general base that accepts a proton from the 2-hydroxyl group of either substrate to facilitate hydride transfer. A second enzymatic group is also required protonated for optimum binding of substrates and may also function as a general acid to donate a proton to the enolpyruvate intermediate to form pyruvate. The (13)C isotope effect, measured on the decarboxylation of D-malate using NAD(+) as the dinucleotide substrate, decreases from a value of 1.0096 +/- 0.0006 with D-malate to 1.00787 +/- 0.00006 with D-malate-2-d, suggesting a stepwise mechanism for the oxidative decarboxylation of D-malate. Using thio-NAD(+) as the dinucleotide substrate the (13)C isotope effects are 1.0034 +/- 0.0007 and 1.0027 +/- 0.0002 with D-malate and D-malate-2-d, respectively.     

Use of primary deuterium and 15N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases

We have used deuterium and 15N isotope effects to study the relative rates of the steps in the mechanisms of alanine and glutamate dehydrogenases. The proposed chemical mechanisms for these enzymes involve carbinolamine formation, imine formation, and reduction of the imine to the amino acid [Grimshaw, C.E., Cook, P.F., & Cleland, W.W. (1981) Biochemistry 20, 5655; Rife, J.E., & Cleland, W.W. (1980) Biochemistry 19, 2328]. These steps are almost equally rate limiting for V/Kammonia with alanine dehydrogenase, while with glutamate dehydrogenase carbinolamine formation, imine formation, and release of glutamate after hydride transfer provide most of the rate limitation of V/Kammonia. Release of oxidized nucleotide is largely rate limiting for Vmax for both enzymes. When beta-hydroxypyruvate replaces pyruvate, or 3-acetylpyridine NADH (Acpyr-NADH) or thio-NADH replaces NADH with alanine dehydrogenase, nucleotide release no longer limits Vmax, and hydride transfer becomes more rate limiting. With glutamate dehydrogenase, replacement of alpha-ketoglutarate by alpha-ketovalerate makes hydride transfer more rate limiting. Use of Acpyr-NADPH has a minimal effect with alpha-ketoglutarate but causes an 8-fold decrease in Vmax with alpha-ketovalerate, with hydride transfer the major rate-limiting step. In contrast, thio-NADPH with either alpha-keto acid causes carbinolamide formation to become almost completely rate limiting. These studies show the power of multiple isotope effects in deducing details of the chemistry and changes in rate-limiting step(s) in complicated reaction mechanisms such as those of alanine and glutamate dehydrogenases.     

Carbinolamine formation and dehydration in a DNA repair enzyme active site

In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1) (27.2 kJ mol(-1)) for the carbinolamine formation reaction and 44.5 kcal mol(-1) (186 kJ mol(-1)) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1) (62.8 kJ mol(-1)) in the forward (formation) reaction and 19 kcal mol(-1) (79.5 kJ mol(-1)) for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N-terminal amine.     

Mechanism of action of Drosophila melanogaster alcohol dehydrogenase.

Reported kinetic pH dependence data for alcohol dehydrogenase from Drosophila melanogaster are analyzed with regard to differences in rate behaviour between this non-metallo enzyme and the zinc-containing liver alcohol dehydrogenase present in vertebrates. For the Drosophila enzyme a mechanism of action is proposed according to which catalytic proton release to solution during alcohol oxidation occurs at the binary-complex level as an obligatory step preceding substrate binding. Such proton release involves an ionizing group with a pKa of about 7.6 in the enzyme.NAD+ complex, tentatively identified as a tyrosyl residue. The ionized form of this group is proposed to participate in the binding of alcohol substrates and to act as a nucleophilic catalyst of the subsequent step of hydride ion transfer from the bound alcohol to NAD+. Herein lie fundamental mechanistic differences between the metallo and non-metallo short chain alcohol dehydrogenases.     

Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 1. Liver alcohol dehydrogenase with benzyl alcohol and yeast aldehyde dehydrogenase with benzaldehyde

Primary intrinsic deuterium and 13C isotope effects have been determined for liver (LADH) and yeast (YADH) alcohol dehydrogenases with benzyl alcohol as substrate and for yeast aldehyde dehydrogenase (ALDH) with benzaldehyde as substrate. These values have also been determined for LADH as a function of changing nucleotide substrate. As the redox potential of the nucleotide changes from -0.320 V with NAD to -0.258 V with acetylpyridine-NAD, the product of primary and secondary deuterium isotope effects rises from 4 toward 6.5, while the primary 13C isotope effect drops from 1.025 to 1.012, suggesting a trend from a late transition state with NAD to one that is more symmetrical. The values of Dk (again the product of primary and secondary isotope effects) and 13k for YADH with NAD are 7 and 1.023, suggesting for this very slow reaction a more stretched, and thus symmetrical, transition state. With ALDH and NAD, the primary 13C isotope effect on the hydride transfer step lies in the range 1.3-1.6%, and the alpha-secondary deuterium isotope effect on the same step is at least 1.22, but 13C isotope effects on formation of the thiohemiacetal intermediate and on the addition of water to the thio ester intermediate are less than 1%. On the basis of the relatively large 13C isotope effects, we conclude that carbon motion is involved in the hydride transfer steps of dehydrogenase reactions.     

Effect of pH on the liver alcohol dehydrogenase reaction.

New transient kinetic methods, which allow kinetics to be carried out under conditions of excess substrate, have been employed to investigate the kinetics of hydride transfer from NADH to aromatic aldehydes and from aromatic alcohols to NAD+ as a function of pH. The hydride transfer rate from 4-deuterio-NADH to beta-naphthaldehyde is nearly pH independent from pH 6.0 to pH 9.9; the isotope effect is also pH independent with kappa-H/kappaD congruent to 2.3. Likewise, the rate of oxidation of benzyl alcohol by NAD+ changes little with pH between pH 8.75 and pH 5.9; the isotope effect for this process is between 3.0 and 4.4. Earlier substituent effect studies on the reduction of aromatic aldehydes were consistent with electrophilic catalysis by either zinc or a protonic acid. The pH independence of hydride transfer is consistent with electrophilic catalysis by zinc since such catalysis by protonic acid (with a pK between 6.0 and 10.0) would show strong pH dependence. However, protonic acid catalysis cannot be excluded if the pKa of the acid catalyst in the ternary NADH-E-RCOH complex were smaller than 6.0 or smaller than 10.0. The two kinetic parameters changing significantly with pH are the kinetic binding constant for ternary complex formation with aromatic alcohol and the rate of dissociation of aromatic alcohols from enzyme. This is consistent with base-catalyzed removal of a proton from alcohol substrated and consequent acid catalysis of protonation of a zinc-alcoholate complex. The equilibrium constant for hydride transfer from benzaldehyde to benzyl alcohol at pH 8.75 is K-eq equals kappa-H/kappa-H equals 42; this constant has important consequences concerning subunit interactions during liver alcohol dehydrogenase catalysis.     

Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding

The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K. Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2. The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction. Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine. These data indicate that the amine substrate must be deprotonated for binding. Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values. Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding. Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue. In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding. A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein. A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate. The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release. A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed. Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.     

Contributions of valine-292 in the nicotinamide binding site of liver alcohol dehydrogenase and dynamics to catalysis

The participation of Val-292 in catalysis by alcohol dehydrogenase and the involvement of dynamics were investigated. Val-292 interacts with the nicotinamide ring of the bound coenzyme and may facilitate hydride transfer. The substitution of Val-292 with Ser (V292S) increases the dissociation constants for the coenzymes (NAD(+) by 50-fold, NADH by 75-fold) and the turnover numbers by 3-7-fold. The V292S enzyme crystallized in the presence of NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol has an open conformation similar to the structure of the wild-type apo-enzyme, rather than the closed conformation observed for ternary complexes with wild-type enzyme. The V292S substitution perturbs the conformational equilibrium of the enzyme and decreases the kinetic complexity, which permits study of the hydride transfer step with steady-state kinetics. Eyring plots show that the DeltaH for the oxidation (V(1)) of the protio and deuterio benzyl alcohols is 13 kcal/mol and that the kinetic isotope effect of 4.1 is essentially temperature-independent. Eyring plots for the catalytic efficiency for reduction of benzaldehyde (V(2)/K(p)) with NADH or NADD are distinctly convex, being temperature-dependent from 5 to 25 degrees C and temperature-independent from 25 to 50 degrees C; the kinetic isotope effect of 3.2 for V(2)/K(p) is essentially independent of the temperature. The temperature dependencies and isotope effects for V(1) and V(2)/K(p) are not adequately explained by semiclassical transition state theory and are better explained by hydride transfer occurring through vibrationally assisted tunneling.     

Supporting role of lysine 13 and glutamate 16 in the acid-base mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase (SDH) catalyzes the NAD+ dependent oxidative deamination of saccharopine to form lysine (Lys) and α-ketoglutarate (α-kg). The active site of SDH has a number of conserved residues that are believed important to the overall reaction. Lysine 13, positioned near the active site base (K77), forms a hydrogen bond to E78 neutralizing it, and contributing to setting the pKa of the catalytic residues to near neutral pH. Glutamate 16 is within hydrogen bond distance to the Nε atom of R18, which has strong H-bonding interactions with the α-carboxylate and α-oxo groups of α-kg. Mutation of K13 to M and E16 to Q decreased kcat by about 15-fold, and primary and solvent deuterium kinetic isotope effects measured with the mutant enzymes indicate hydride transfer is rate limiting for the overall reaction. The pH-rate profiles for K13M exhibited no pH dependence, consistent with an increase in negative charge in the active site resulting in the perturbation in the pKas of catalytic groups. Elimination of E16 affects optimal positioning of R18, which is involved in binding and holding α-kg in the correct conformation for optimum catalysis. In agreement, a ΔΔG°' of 2.60 kcal/mol is estimated from the change in Kα-kg for replacing E16 with Q.     

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.