Modification of a thiol at the active site of the Ascaris suum NAD-malic enzyme results in changes in the rate-determining steps for oxidative decarboxylation of L-malate.

A thiol group at the malate-binding site of the NAD-malic enzyme from Ascaris suum has been modified to thiocyanate. The modified enzyme generally exhibits slight increases in K(NAD) and K(i metal) and decreases in V(max) as the metal size increases from Mg2+ to Mn2+ to Cd2+, indicative of crowding in the site. The K(malate) value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/K(malate) increase with all three metal ions compared to the native enzyme concomitant with a decrease in the C-13 isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The C-13 effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary C-13 effect in the hydride transfer step, similar to data obtained with non-nicotinamide-containing dinucleotide substrates for the native enzyme (see the preceding paper in this issue). The native enzyme is inactivated in a time-dependent manner by Cd2+. This inactivation occurs whether the enzyme alone is present or whether the enzyme is turning over with Cd2+ as the divalent metal activator. Upon inactivation, only Cd2+ ions are bound at high stoichiometry to the enzyme, which eventually becomes denatured. Conversion of the active-site thiol to thiocyanate makes it more difficult to inactivate the enzyme by treatment with Cd2+.