SITE-DIRECTED MUTAGENESIS AS A PROBE OF NAD+-BINDING SITE IN MALIC ENZYME

Abstract

The mitochondrial NAD-malic enzyme catalyzes the oxidative decarboxylation of malate to pyruvate and CO2. The role of the dinucleotide substrate in oxidative decarboxylation is probed in this study using site-directed mutagenesis to change key residues that line the dinucleotide binding site. Mutant enzymes were characterized using initial rate kinetics, and isotope effects were used to obtain information on the contribution of these residues to binding energy and catalysis.

The first part of the project was to investigate the contribution of binding energy and catalysis of the groups that interact with the nicotinamide and ribose rings of NAD. Results obtained for the N479 mutant enzymes, indicate that the hydrogen bond donated by N479 to the carboxamide side chain of the nicotinamide ring is important for proper orientation of the cofactor in the hydride transfer step. The stepwise oxidative decarboxylation mechanism observed for the wt enzyme changed to a concerted one, which is totally rate limiting, for the N479Q mutant enzyme. In this case, it is likely that the longer glutamine side chain causes reorientation of malate such that it binds in a conformation that is optimal for concerted oxidative decarboxylation. Converting N479 to the shorter serine side chain gives very similar values of KNAD, Kmalate and isotope effects relative to wt, but V/Et is decreased 2,000-fold. Data suggest an increased freedom of rotation, resulting in nonproductively bound cofactor, perhaps with the nicotinamide ring occupying the site that favors the reduced ring. Changes were also made to two residues, S433 and N434, which interact with the nicotinamide ribose of NAD. In addition, N434 donates a hydrogen bond to the β-carboxylate of malate. The KNAD for the S433A mutant enzyme increased by 80-fold, indicating that this residue provides significant binding affinity for the dinucleotide. With N434A, the interaction of the residue with malate is lost, causing the malate to reorient itself, leading to a slower decarboxylation step. The longer glutamine and methionine side chains stick into the active site and cause a change in the position of malate and/or NAD, and results in more than a 104-fold decrease in V/Et for these mutant enzymes. Overall, data indicate that subtle changes in the orientation of the cofactor and substrate dramatically influence the reaction rate.

The second part of this project focused on the residues that form the adenosine binding site of NAD. Site-directed mutagenesis was performed to determine the role of these residues in binding of the cofactor and/or catalysis. D361, which is completely conserved among species, is located in the dinucleotide-binding Rossmann fold and makes a salt bridge with R370, which is also highly conserved. D361 was mutated to E, A and N. R370 was mutated to K and A. D361E and A mutant enzymes were inactive, likely a result of the increase in the volume, in the case of the D361E mutant enzyme that caused clashes with the surrounding residues, and loss of the ionic interaction between D361 and R370, for D361A. Although the Km for the substrates and isotope effect values did not show significant changes for the D361N mutant enzyme, V/Et decreased by 1400-fold. Data suggested the nonproductive binding of the cofactor, giving a low fraction of active enzyme. The R370K mutant enzyme did not show any significant changes in the kinetic parameters, while the R370A mutant enzyme gave a slight change in V/Et, contrary to expectations. Overall, results suggest that the salt bridge between D361 and R370 is important for maintaining the productive conformation of the NAD binding site. Mutation of residues involved leads to nonproductive binding of NAD. The interaction stabilizes one of the Rossmann fold loops that NAD binds. Mutation of H377 to lysine, which is conserved in NADP-specific malic enzymes and proposed to be a cofactor specificity determinant, did not cause a shift in cofactor specificity of the Ascaris malic enzyme from NAD to NADP. However, it is confirmed that this residue is an important second layer residue that affects the packing of the first layer residues that directly interact with the cofactor.

The last part of this dissertation consists of a review on the acid-base chemical mechanism of the enzyme class, metal ion-dependent pyridine nucleotide-linked β-hydroxyacid oxidative decarboxylases. This family includes malic enzyme (ME), isocitrate dehydrogenase (IcDH), and isopropylmalate dehydrogenase (IPMDH), which require a divalent metal ion, and homoisocitrate dehydrogenase (HIcDH), and tartrate dehydrogenase (TDH), which require a monovalent and divalent metal ion for activity. Overall structure gives two distinct classes, with the MEs as the only member of one of the two classes, ME subfamily, while all of the others exhibit the same fold, the IcDH subfamily. The active sites of all of the enzymes have a similar overall geometry and most of the active site residues are conserved throughout the family; they are completely conserved within the IcDH subfamily. Data available for all of the enzymes in the family have been considered and a general mechanism is proposed for the family that makes use of a lysine (general base), tyrosine (general acid) pair. Differences exist in the mechanism of generating the neutral form of lysine so that it can act as a base.