Stepwise versus Concerted Oxidative Decarboxylation Catalyzed by Malic Enzyme: A Reinvestigation

The NAD-malic enzyme catalyzes the divalent metal-ion-dependent oxidative decarboxylation of L-malate to yield CO2, pyruvate, and the reduced dinucleotide. With Mg2+ as the divalent metal ion activator, primary deuterium and tritium isotope effects have been obtained with several different alternati...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-03, Vol.33 (8), p.2096-2103
Main Authors: Karsten, William E, Cook, Paul F
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Analytical, structural and metabolic biochemistry
Animals
Ascaris
ASCARIS SUUM
Biological and medical sciences
Carboxylic Acids - metabolism
Catalysis
Chickens
COENZIMAS
COENZYME
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Horses
Isotopes
MAGNESIO
MAGNESIUM
Malate Dehydrogenase - isolation & purification
Malate Dehydrogenase - metabolism
NAD - analogs & derivatives
NAD - metabolism
NADP - metabolism
NUCLEOTIDE
NUCLEOTIDOS
Oxaloacetates
Oxidation-Reduction
Oxidoreductases
OXIDORREDUCTASAS
OXYDOREDUCTASE
REACCIONES QUIMICAS
REACTION CHIMIQUE
SECUENCIA NUCLEICA
SEQUENCE NUCLEIQUE
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Summary:The NAD-malic enzyme catalyzes the divalent metal-ion-dependent oxidative decarboxylation of L-malate to yield CO2, pyruvate, and the reduced dinucleotide. With Mg2+ as the divalent metal ion activator, primary deuterium and tritium isotope effects have been obtained with several different alternative dinucleotide substrates. The partitioning ratio of oxalacetate to malate and pyruvate has also been determined with either NAD or 3-acetyl pyridine adenine dinucleotide (3-APAD). These data have been used to calculate estimates of commitment factors and intrinsic isotope effects for the NAD-malic enzyme reaction. The calculated values of the intrinsic 13C and deuterium isotope effects with NADP are similar to the previously determined values for the chicken liver malic enzyme (Grissom, C.B., and Cleland, W.W. (1988) Biochemistry 27, 2927) and suggest that the transition-state structures are similar for the Ascaris NAD- and chicken liver NADP-malic enzymes. With NAD or NADP as the dinucleotide substrate, the data are all consistent with a stepwise chemical mechanism with oxidation of L-malate at C2 preceding decarboxylation of the bound oxalacetate intermediate. However, none of the data with the alternative dinucleotide substrates, 3-acetylpyridine adenine dinucleotide and 3-pyridine aldehyde adenine dinucleotide (PAAD), can be fit with satisfaction to the various criteria that support a stepwise mechanism with NAD(P). The mechanism with 3-APAD and PAAD is likely concerted. The most likely explanation for a change in the mechanism for oxidative decarboxylation from stepwise with NAD(P) to concerted with alternative dinucleotide substrates such as 3-APAD and PAAD is a difference in the configuration of bound malate when the different dinucleotide substrates are used
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00174a016