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 KNAD and Ki metal and decreases in Vmax as the metal size increases from Mg2+ to Mn2+ to Cd2+, indicative of crowding in the site. The Kmalate value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/Kmalate increase with all three metal ions compared to the native enzyme concomitant with a decrease in the 13C isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The 13C effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary 13C 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+.
Gavva
S R
SR
Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine, Ft. Worth 76107.
Harris
B G
BG
Weiss
P M
PM
Cook
P F
PF
D000818:Animals; D001200:Ascaris / Q000187:drug effects / Q000201:enzymology*; D001665:Binding Sites / Q000187:drug effects; D002104:Cadmium / Q000494:pharmacology; D002247:Carbon Isotopes; D003653:Decarboxylation; D003903:Deuterium; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000737:chemistry*; D008293:Malates / Q000737:chemistry / Q000378:metabolism*; D008345:Manganese / Q000494:pharmacology; D013861:Thiocyanates / Q000737:chemistry*
D002247:Carbon Isotopes; D008293:Malates; D013861:Thiocyanates; D002104:Cadmium; D008345:Manganese; C030298:malic acid; D003903:Deuterium; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); D008274:Magnesium; C031760:thiocyanate