Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction.
Deuterium isotope effects and 13C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the 13C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. When dinucleotide substrates such as thio-NAD, 3-acetylpyridine adenine dinucleotide, and 3-pyridinealdehyde adenine dinucleotide that contain modified nicotinamide rings are used, the 13C effect increases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the 13C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a beta-secondary 13C isotope effect accompanies hydride transfer as a result of hyperconjugation of the beta-carboxyl of malate as the transition state for the hydride transfer step is approached.(ABSTRACT TRUNCATED AT 250 WORDS)
Weiss
P M
PM
Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine, Ft. Worth 76107.
Gavva
S R
SR
Harris
B G
BG
Urbauer
J L
JL
Cleland
W W
WW
Cook
P F
PF
D000818:Animals; D001200:Ascaris; D002247:Carbon Isotopes; D002645:Chickens; D003653:Decarboxylation; D003903:Deuterium; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D009243:NAD / Q000378:metabolism* / Q000031:analogs & derivatives; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D002247:Carbon Isotopes; D008293:Malates; D009243:NAD; C003451:thionicotinamide adenine dinucleotide; D009249:NADP; C030298:malic acid; C005941:3-acetylpyridine adenine dinucleotide; D003903:Deuterium; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)