Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

Journal Article · Tue Jun 11 04:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00237a018· OSTI ID:5026009

Weiss, P M; Urbauer, J L; Cleland, W W ^[1]; Gavva, S R; Harris, B G; Cook, P F ^[2]

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1. Univ. of Wisconsin, Madison (United States)
2. Texas Coll. of Osteopathic Medicine, Ft. Worth (United States)

Deuterium isotope effects and {sup 13}C 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 {sup 13}C 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. When dinucleotide substrates such as thio-NAD, 3-nicotinamide rings are used, the {sup 13}C 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 {sup 13}C 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 {sup 13}C 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.

• OSTI ID: 5026009
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:23; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CARBON 13
CARBON ISOTOPES
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
COENZYMES
DECARBOXYLASES
DECARBOXYLATION
DEUTERIUM COMPOUNDS
ENZYMES
EVEN-ODD NUCLEI
HYDROGEN COMPOUNDS
HYDROXY ACIDS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LIGHT NUCLEI
LYASES
MALIC ACID
NAD
NADP
NUCLEI
NUCLEOTIDE DEHYDROGENASES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
REACTION KINETICS
REDOX POTENTIAL
STABLE ISOTOPES
SUBSTRATES