PH dependence of deuterium isotope effects and tritium exchange in the bovine plasma amine oxidase reaction: a role for single-base catalysis in amine oxidation and imine exchange
The pH dependence of steady-state parameters for (1,1-1H2)- and (1,1-2H2)benzylamine oxidation and of tritium exchange from (2-/sup 3/H)dopamine has been measured in the bovine plasma amine oxidase reaction. Deuterium isotope effects on kcat/Km for benzylamine are observed to be constant, near the intrinsic value of 13.5, over the experimental pH range, indicating that C-H bond cleavage is fully rate limiting for this parameter. As a consequence, pKa values derived from kcat/Km profiles, 8.0 +/- 0.1 (pK1) and 9.0 +/- 0.16 (pKs), can be ascribed to microscopic pKa values for the ionization of an essential active site residue (EB1) and substrate, respectively. Profiles for kcat and Dkcat show that EB1 undergoes a perturbation from 8.0 to 5.6 +/- 0.3 (pK1') in the presence of substrate; additionally, a second ionization, pK2 = 7.25 +/- 0.25, is observed to mediate but not be essential for enzyme reoxidation. The pH dependence of the ratio of tritium exchange to product formation for dopamine also indicates base catalysis with a pKexch = 5.5 +/- 0.01, which is within experimental error of pK1'. We conclude that the data presented herein support a single residue catalyzing both substrate oxidation and exchange, consistent with recent stereochemical results that implicate a syn relationship between these processes (Farnum, M., and Klinman, J.P. (1985) Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1055). This conclusion contrasts with earlier kinetic data in support of a large rate differential for the exchange of hydrogen from C-1 vs. C-2 of phenethylamine derivatives.
- Research Organization:
- Univ. of California, Berkeley
- OSTI ID:
- 5207495
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 8; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
AMINES
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARDIOTONICS
CARDIOVASCULAR AGENTS
CATALYSIS
CATTLE
CHEMICAL REACTIONS
DEUTERIUM
DOMESTIC ANIMALS
DOPAMINE
DRUGS
ENZYME ACTIVITY
ENZYMES
HYDROGEN ISOTOPES
HYDROXY COMPOUNDS
IMINES
ISOTOPE EFFECTS
ISOTOPES
ISOTOPIC EXCHANGE
KINETICS
LIGHT NUCLEI
MAMMALS
NEUROREGULATORS
NUCLEI
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDATION
OXIDOREDUCTASES
PH VALUE
PHENOLS
POLYPHENOLS
RADIOISOTOPES
REACTION KINETICS
RUMINANTS
STABLE ISOTOPES
SYMPATHOMIMETICS
TRITIUM
VERTEBRATES
YEARS LIVING RADIOISOTOPES