Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide malic enzyme reaction from isotope effects and pH studies

Abstract

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.

Crystallographic Studies on Ascaris suum NAD-Malic Enzyme Bound to Reduced Cofactor and Identification of an Effector Site

“…Implications to Catalytic Mechanism-A three-step acidbase catalytic mechanism is proposed for ascarid malic enzyme based on pH and isotope partitioning studies (25)(26)(27). The assignment of the general acid, general base, and binding groups shown in Scheme 1 is based on the x-ray structure of the ME⅐NAD complex (11) and site-directed mutagenesis studies (19).…”

On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis

“…However, ionizable groups involved with these steps will not be seen in the k cat pH-rate profiles if binding occurs only when all ionizable groups are in the correct protonation state when they fall outside the range analyzed, or when a conformational change is rate-limiting [15]. Although unusual, a similar pH-dependence of k cat , from pH 5.5 to 9.5, was reported for A. suum NAD + -dependent malic dehydrogenase-catalyzed oxidative decarboxylation of malate [32]. In the case of GabD1, it seems rational to suggest that the carboxylate from Glu285 cannot be seen, as its p K a value is probably below 5.…”

Secondary 18O and Primary 13C Isotope Effects as a Probe of Transition-State Structure for Enzymic Decarboxylation of Oxalacetate

“…The pH dependence of oxalate binding to the enzyme is consistent with this assignment for the enzyme residue. It should be noted that the pH dependence of both Vand V/Kfor OAA decarboxylation by NAD-malic enzyme (Kiick et al, 1986;Park et al, 1986) exhibits the same characteristics as OAA decarboxylase.…”