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

Journal Article · Tue Apr 22 00:00:00 EST 1986 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00356a010· OSTI ID:5207495

Farnum, M; Palcic, M; Klinman, J P

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), Vol. 8
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC
ORGANIC
PHYSICAL AND ANALYTICAL CHEMISTRY
AMINES
CATALYSIS
DEUTERIUM
ISOTOPE EFFECTS
TRITIUM
ISOTOPIC EXCHANGE
BIOCHEMICAL REACTION KINETICS
CATTLE
DOPAMINE
ENZYME ACTIVITY
IMINES
OXIDATION
OXIDOREDUCTASES
PH VALUE
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARDIOTONICS
CARDIOVASCULAR AGENTS
CHEMICAL REACTIONS
DOMESTIC ANIMALS
DRUGS
ENZYMES
HYDROGEN ISOTOPES
HYDROXY COMPOUNDS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
NEUROREGULATORS
NUCLEI
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHENOLS
POLYPHENOLS
RADIOISOTOPES
REACTION KINETICS
RUMINANTS
STABLE ISOTOPES
SYMPATHOMIMETICS
VERTEBRATES
YEARS LIVING RADIOISOTOPES
550201* - Biochemistry- Tracer Techniques
400302 - Organic Chemistry- Isotope Effects- (-1987)