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

• Published in: Biochemistry (Easton) 1986-04, Vol.25 (8), p.1898-1904
• Main Authors: Farnum, Martin, Palcic, Monica, Klinman, Judith Pollock
• Format: Article
• Language: English
• Subjects: Amine Oxidase (Copper-Containing); amine oxidase(flavin-containing); Analytical, structural and metabolic biochemistry; Animals; benzylamine; Binding Sites; Biological and medical sciences; Cattle; Deuterium; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Kinetics; Mathematics; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on CH-NH Group Donors - blood; plasma; Radioisotope Dilution Technique
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00356a010

The pH dependence of steady-state parameters for [1,1-1H2]- and [1,1-2H2]benzylamine oxidation and of tritium exchange from [2-3H]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., & 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 [Palcic, M.M., & Klinman, J.P. (1983) Biochemistry 22, 5957-5966].