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The role of histidine residues in glutamate dehydrogenase.

ABSTRACT: 1. Glutamate dehydrogenase was subject to rapid inactivation when irradiated in the presence of Rose Bengal or incubated in the presence of ethoxyformic anhydride. 2. Inactivation in the presence of Rose Bengal led to the photo-oxidation of four histidine residues. Oxidation of three histidine residues had little effect on enzyme activity, but oxidation of the fourth residue led to the almost total loss of activity. 3. Acylation of glutamate dehydrogenase with ethoxyformic anhydride at pH6.1 led to the modification of three histidine residues with a corresponding loss of half the original activity. Acylation at pH7.5 led to the modification of two histidine residues and a total loss of enzyme activity. 4. One of the histidine residues undergoing reaction at pH6.1 also undergoes reaction at pH7.5. 5. The presence of either glutamate or NAD(+) in the reaction mixtures at pH6.1 had no appreciable effect. At pH7.5 glutamate caused a marked decrease in both the degree of alkylation and degree of inactivation. NAD(+) had no effect on the degree of inactivation at pH7.5 but did modify the extent of acylation. 6. The normal response of the enzyme towards ADP was unaffected by acylation at pH6.1 or 7.5. 7. The normal response of the enzyme towards GTP was altered by treatment at both pH6.1 and 7.5.

SUBMITTER: Tudball N

PROVIDER: S-EPMC1174091 | biostudies-other | 1972 Sep

REPOSITORIES: biostudies-other

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Project description:His-48 in yeast alcohol dehydrogenase I (His 51 in horse liver alcohol dehydrogenase) is a highly conserved residue in the active sites of many alcohol dehydrogenases. The imidazole group of His-48 may participate in base catalysis of proton transfer as it is linked by hydrogen bonds through the 2'-hydroxyl group of the nicotinamide ribose and the hydroxyl group of Thr-45 to the hydroxyl group of the alcohol bound to the catalytic zinc. In this study, His-48 was substituted with a glutamic acid residue to determine if a carboxylate could replace imidazole or to a serine residue to determine if the exposure of the 2'-hydroxyl group of the ribose to solvent would allow proton transfer to water without base catalysis. At pH 7.3, the H48E substitution increases affinity for NAD+ and NADH 17- or 2.6-fold, but decreases catalytic efficiency (V/Km) on ethanol by 70-fold and on acetaldehyde by 6-fold relative to wild-type enzyme. The H48S substitution increases affinity for coenzymes by 2-fold and decreases (V/Km) on ethanol and acetaldehyde only by ∼3-fold. The substituted enzymes show substrate deuterium isotope (H/D) effects of 3-4 for turnover number (V1) and catalytic efficiency (V1/Kb) for ethanol oxidation, indicating that hydrogen transfer is partially rate-limiting and suggesting a somewhat more random mechanism for binding of ethanol and NAD+. For reduction of acetaldehyde, the deuterium isotope effects are small, and the kinetic mechanism appears to be ordered for binding of NADH first and acetaldehyde next. The pH dependencies for H48E and H48S ADHs can be described by a mechanism with pK values of about 6-7 and 9. However, the pH dependencies for oxidation of ethanol and butanol by the H48S enzyme are also simply described by a straight line, with slopes of log V1/Kb against pH of 0.37 or 0.43, respectively. The linear dependence apparently represents catalysis by hydroxide that has a low activity coefficient due to the protein environment, or to a kinetically complex proton transfer. The effects of the substitutions of His-48 show that this residue contributes to catalysis, although many dehydrogenases also have other residues.

Project description: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 Å 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 Å, 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 V₂/K(Lys)E(t) and V₂/K(α-kg)E(t) (the double mutation gives >10⁵-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 V₂/K(Lys). A value of 2.0 was also observed for (D)(V₂/K(Lys))(D₂O) when the primary deuterium kinetic isotope effect was repeated in D₂O, consistent with a rate-limiting hydride transfer step. A viscosity effect of 0.8 was observed on V₂/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, V₂/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 H₂O or D₂O, 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 V₂/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.

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The role of histidine residues in glutamate dehydrogenase.

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