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

Abstract

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].(ABSTRACT TRUNCATED AT 250 WORDS)

Human kidney diamine oxidase: heterologous expression, purification, and characterization

“…The higher pK a likely represents the ionization of the product Schiff base (Scheme 1, species B, C). Klinman and co-workers [36,37] have described detailed work on the pH dependence of steady-state parameters for the CAOs from bovine plasma and a yeast, and our data are consistent with their interpretation. A plot of 1/K M versus pH reflects ionization potentials in the free enzyme and free substrate [29].…”

“…Detailed kinetic studies have elucidated many of the details of the reductive half-reaction. Proton abstraction from the substrate Schiff base (species B) has been shown to at least partially contribute to the rate-determining step in some mammalian CAOs [36,75]. Substrate turnover (k cat ) values for rhKDAO are quite similar among the substrates investigated in this work, from 1 to 10 s -1 , suggesting a common rate-limiting step.…”

Multiple Binding Sites for Substrates and Modulators of Semicarbazide-Sensitive Amine Oxidases: Kinetic Consequences

“…In designating ES to represent both E ox -NH ϩ CH 2 R and E red , we imply the existence of only one enzyme species (E), with formation of Schiff's base intermediates, release of aldehyde, and generation of an aminoquinol (Mure et al, 2002), occurring instantaneously. The rate-limiting contribution of this reductive half-reaction (Farnum et al, 1986) indicates that this implication is incorrect. We have nevertheless found these equations useful for fitting multiple data sets with the global nonlinear regression facility of GraphPad Prism, because acceptable global fits are generally obtained only when underlying models are correct.…”

New Quinocofactors in Eukaryotes

“…Second, the likely active site base is seen to be an Asp that sits opposite the copper in relation to the cofactor. This Asp is found in a highly hydrophobic environment, which may explain the elevated pK a ascribed to the active site base in the resting form of bovine serum amine oxidase (pK a ϭ 7.8, reduced to pK a ϭ 5.3 once substrate has bound (27)). There is sufficient room within the protein active site for the TPQ ring to rotate, an essential feature of the biogenetic mechanism shown in Scheme 1.…”