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.     

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.     

Crystal structures of ligand-bound saccharopine dehydrogenase from Saccharomyces cerevisiae

Three structures of saccharopine dehydrogenase (l-lysine-forming) (SDH) have been determined in the presence of sulfate, adenosine monophosphate (AMP), and oxalylglycine (OxGly). In the sulfate-bound structure, a sulfate ion binds in a cleft between the two domains of SDH, occupies one of the substrate carboxylate binding sites, and results in partial closure of the active site of the enzyme due to a domain rotation of almost 12 degrees in comparison to the apoenzyme structure. In the second structure, AMP binds to the active site in an area where the NAD+ cofactor is expected to bind. All of the AMP moieties (adenine ring, ribose, and phosphate) interact with specific residues of the enzyme. In the OxGly-bound structure, carboxylates of OxGly interact with arginine residues representative of the manner in which substrate (alpha-ketoglutarate and saccharopine) may bind. The alpha-keto group of OxGly interacts with Lys77 and His96, which are candidates for acid-base catalysis. Analysis of ligand-enzyme interactions, comparative structural analysis, corroboration with kinetic data, and discussion of a ternary complex model are presented in this study.     

Complete kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated to be 11 +/- 2 mM as measured using Mg2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg2+ and alpha-ketoadipate. Curvature is observed in the double-reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases, the inhibition changes to uncompetitive, consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO2, alpha-ketoadipate, and NADH. Isocitrate is a slow substrate with a rate (V/E(t)) 216-fold slower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg2+ bind to the enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The Keq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.     

Determinants of substrate specificity for saccharopine dehydrogenase from Saccharomyces cerevisiae

A survey of NADH, alpha-Kg, and lysine analogues has been undertaken in an attempt to define the substrate specificity of saccharopine dehydrogenase and to identify functional groups on all substrates and dinucleotides important for substrate binding. A number of NAD analogues, including NADP, 3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), and thionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deamination reaction, as can a number of alpha-keto analogues, including glyoxylate, pyruvate, alpha-ketobutyrate, alpha-ketovalerate, alpha-ketomalonate, and alpha-ketoadipate. Inhibition studies using nucleotide analogues suggest that the majority of the binding energy of the dinucleotides comes from the AMP portion and that distinctly different conformations are generated upon binding of the oxidized and reduced dinucleotides. Addition of the 2'-phosphate as in NADPH causes poor binding of subsequent substrates but has little effect on coenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparison to NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studies using aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acid binding pocket. Data suggest that a side chain with three carbons (from the alpha-keto group up to and including the side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5 carboxylate of the alpha-keto acid is also important for binding; the alpha-oxo group contributes a factor of 10 to affinity. The keto acid binding pocket is relatively large and flexible and can accommodate the bulky aromatic ring of a pyridine dicarboxylic acid and a negative charge at the C3 but not the C4 position. However, the amino acid binding site is hydrophobic, and the optimal length of the hydrophobic portion of the amino acid carbon side chain is three or four carbons. In addition, the amino acid binding pocket can accommodate a branch at the gamma-carbon, but not at the beta-carbon.     

Purification and properties of saccharopine dehydrogenase (glutamate forming) in the Saccharomyces cerevisiae lysine biosynthetic pathway.

Saccharopine dehydrogenase (glutamate forming) of the biosynthetic pathway of lysine in Saccharomyces cerevisiae was purified 1,122-fold by using acid precipitation, ammonium sulfate precipitation, DEAE-Sepharose, gel filtration, and Reactive Red-120 agarose chromatography. The enzyme exhibited a native molecular size of 69,000 daltons by gel filtration and consisted of a single 50,000-dalton polypeptide based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was readily denatured by exposures to temperatures exceeding 46 degrees C. The pH optimum for the reverse reaction was 9.5. The apparent Kms for L-saccharopine and NAD+ were 2.32 and 0.054 mM, respectively. The enzyme was inhibited by mercuric chloride but not by carbonyl or metal complexing agents.     

Homoserine dehydrogenase from Saccharomyces cerevisiae: kinetic mechanism and stereochemistry of hydride transfer

Homoserine dehydrogenase (HSD), which is required for the synthesis of threonine, isoleucine and methionine in fungi, is a potential target for novel antifungal drugs. In order to design effective inhibitors, the kinetic mechanism of Saccharomyces cerevisiae HSD and the stereochemistry of hydride transfer were examined. Product inhibition experiments revealed that yeast HSD follows an ordered Bi Bi kinetic mechanism, where NAD(P)H must bind the enzyme prior to aspartate semialdehyde (ASA) and homoserine is released first followed by NAD(P)+. H-(1,2,4-triazol-3-yl)-D,L-alanine was an uncompetitive inhibitor of HSD with respect to NADPH (K(ii)=3.04+/-0.18 mM) and a noncompetitive inhibitor with respect to ASA (K(is)=1.64+/-0.36 mM, K(ii)=3.84+/-0.46 mM), in agreement with the proposed substrate order. Both kinetic isotope and viscosity experiments provided evidence for a very rapid catalytic step and suggest nicotinamide release to be primarily rate limiting. Incubation of HSD with stereospecifically deuterated NADP[2H] and subsaturating amounts of aspartate semialdehyde revealed that the pro-S NADPH hydride is transferred to the aldehyde. The pH dependence of steady state kinetic parameters indicate that ionizable groups with basic pKs may be involved in substrate binding, consistent with the observation of Lys223 at the enzyme active site in the recently determined 3D structure [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. These findings provide the requisite foundation for future exploitation of fungal HSD in inhibitor design.     

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.     

Binding studies of NADPH to NADP-specific L-glutamate dehydrogenase from Saccharomyces cerevisiae.

Optical characteristics of enzyme-reduced coenzyme complexes of yeast NADP-specific glutamate dehydrogenase have been investigated in the presence and absence of product (L-glutamate) and in the presence or absence of phosphate. The phosphate effect, pointed out in a previous work, is found again: inorganic phosphate (Pi) destabilizes the binary complex (E - NADPH), the dissociation constant of which is equal to 14 muM, a value much higher than that determined in Tris-HCl buffer: Kd = 0.9 muM. Concerning the role of phosphate some assumptions are drawn up with respect to a similar behaviour of Pi toward yeast glutamate dehydrogenase and ADP toward the beef liver enzyme. In the same way, L-glutamate induces a stabilization of the binary complex; this latter effect is unchanged in the presence of phosphate, yet it is less marked than in the case of beef liver glutamate dehydrogenase. Protein fluorescence, nucleotide fluorescence and circular dichroism measurements allowed the determination of three identical and independent NADPH binding sites per hexameric active unit. In analogy with beef liver enzyme, it seems that yeast glutamate dehydrogenase is a good model to study anticooperativity in ligand binding.     

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.     

Role of arginine residue in saccharopine dehydrogenase (L-lysine forming) from baker's yeast

The baker's yeast saccharopine dehydrogenase (EC 1.5.1.7) was inactivated by 2,3-butanedione following pseudo-first-order reaction kinetics. The pseudo-first-order rate constant for inactivation was linearly related to the butanedione concentration, and a value of 7.5 M-1 min-1 was obtained for the second-order rate constant at pH 8.0 and 25 degrees C. Amino acid analysis of the inactivated enzyme revealed that arginine was the only amino acid residue affected. Although as many as eight arginine residues were lost on prolonged incubation with butanedione, only one residue appears to be essential for activity. The modification resulted in the change in Vmax, but not in Km, values for substrates. The inactivation by butanedione was substantially protected by L-leucine, a competitive analogue of substrate lysine, in the presence of reduced nicotinamide adenine dinucleotide (NADH) and alpha-ketoglutarate. Since leucine binds only to the enzyme-NADH-alpha-ketoglutarate complex, the result suggests that an arginine residue located near the binding site for the amino acid substrate is modified. Titration with leucine showed that the reaction of butanedione also took place with the enzyme-NADH-alpha-ketoglutarate-leucine complex more slowly than with the free enzyme. The binding study indicated that the inactivated enzyme still retained the capacity to bind leucine, although the affinity appeared to be somewhat decreased. From these results it is concluded that an arginine residue essential for activity is involved in the catalytic reaction rather than in the binding of the coenzyme and substrates.     

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.     

Kinetics and reaction mechanism of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae.

The reaction of solubilized cytochrome oxidase in the fully reduced state with O2 at low temperatures reveals components with characteristics similar to those observed with the membrane-bound oxidase, namely compounds A and B, which are proposed to be 'oxy' and 'peroxy' compounds respectively. Similar species are identified in both solubilized and membrane-bound oxidases; the reaction velocity constant for the reation with O2 and the dissociation constant are decreased 2-3-fold in the solubilied preparation as compared with the membrane-bound species, owing to decreased reactivity towards O2 in the former. The oxidase prepared in the mixed-valence state shows the distinctive absorption band characteristic of compound C, identified in the membrane-bound oxidase. The assignment of the alpha, beta, gamma and near-i.r. absorption bands to possible valence states of these compounds is made.     

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.     

Enzymatic measurement of saccharopine with saccharopine dehydrogenase

We have developed an enzymatic method for measuring saccharopine, a key intermediate in lysine metabolism. With the enzyme saccharopine dehydrogenase, saccharopine can be oxidized to lysine and alpha-ketoglutarate with the corresponding conversion of NAD to NADH. The natural equilibrium favors saccharopine formation, but using hydrazine to trap one of the products, alpha-ketoglutarate, shifts the reaction toward quantitative oxidation of saccharopine. A stable endpoint is reached in 15-20 min, and although high concentrations of alpha-ketoglutarate slow the reaction, the end product is fully recovered. Unlike previous assays this technique is specific, convenient, and capable of measuring saccharopine directly in protein-free biological fluids or extracts.