Use of primary deuterium and /sup 15/N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases

Journal Article · Tue Jun 28 04:00:00 EDT 1988 · Biochemistry; (United States)

OSTI ID:6864099

Weiss, P M; Chen, C Y; Cleland, W W; Cook, P F

The authors have used deuterium and /sup 15/N isotope effects to study the relative rates of the steps in the mechanisms of alanine and glutamate dehydrogenases. The proposed chemical mechanisms for these enzymes involve carbinolamine formation, imine formation, and reduction of the imine to the amino acid. These steps are almost equally rate limiting for V/K/sub ammonia/ with alanine dehydrogenase, while with glutamate dehydrogenase carbinolamine formation, imine formation, and release of glutamate after hydride transfer provide most of the rate limitation of V/K/sub ammonia/. Release of oxidized nucleotide is largely rate limiting for V/sub max/ for both enzymes. When ..beta..-hydroxypyruvate replaces pyruvate, or 3-acetylpyridine NADH (Acpyr-NADH) or thio-NADH replaces NADH with alanine dehydrogenase, nucleotide release no longer limits V/sub max/, and hydride transfer becomes more rate limiting. With glutamate dehydrogenase, replacement of ..cap alpha..-ketoglutarate by ..cap alpha..-ketovalerate makes hydride transfer more rate limiting. Use of Acpyr-NADPH has a minimal effect with ..cap alpha..-ketoglutarate but causes an 8-fold decrease in V/sub max/ with ..cap alpha..-ketovalerate, with hydride transfer the major rate-limiting step. In contrast, thio-NADPH with either ..cap alpha..-keto acid causes carbinolamide formation to become almost completely rate limiting. These studies show the power of multiple isotope effects in deducing details of the chemistry and changes in rate-limiting step(s) in complicated reaction mechanisms such as those of alanine and glutamate dehydrogenases.

• Research Organization: North Texas State Univ., Denton (USA)
• OSTI ID: 6864099
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:13; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ALANINES
AMINO ACIDS
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
DEUTERIUM COMPOUNDS
ENZYMES
GLUTAMIC ACID
HEMIACETAL DEHYDROGENASES
HYDROGEN COMPOUNDS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
NITROGEN 15
NITROGEN ISOTOPES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PH VALUE
REACTION KINETICS
STABLE ISOTOPES
TRITIUM COMPOUNDS