Structure-function studies of substrate oxidation by bovine serum amine oxidase: relationship to cofactor structure and mechanism.
The chemical mechanism of substrate oxidation, catalyzed by bovine serum amine oxidase, has been explored by a detailed investigation of structure-reactivity correlations. Past mechanistic studies, involving the reductive trapping of substrate to cofactor [Hartmann, C., & Klinman, J. P. (1987) J. Biol. Chem. 262, 962], implied the intermediacy of a substrate imine complex in the catalytic redox mechanism. These studies led to the proposal of a transamination mechanism for substrate oxidation, analogous to pyridoxal phosphate dependent enzymes. In pyridoxal phosphate catalyzed reactions, the transamination process involves the transient formation of a resonance-stabilized carbanion intermediate. Although evidence has been presented describing the participation of an active site base in bovine serum amine oxidase catalysis [Farnum, M. F., Palcic, M. M., & Klinman, J. P. (1986) Biochemistry 25, 1898], the nature of the intermediate derived from C-H bond cleavage has not been directly addressed. To examine this question, a structure-reactivity study was performed using a series of para-substituted benzylamines. Having prior knowledge of the intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic rate constants from steady-state data [Palcic, M. M., & Klinman, J. P. (1983) Biochemistry 22, 5957]. Deuterium isotope effects on kcat and kcat/Km parameters were determined for all substrates, allowing for the calculation of rate constants for C-H bond cleavage (k3) and substrate dissociation constants (Kd). Pre-steady-state constants obtained for p-acetylbenzylamine, p-(trifluoromethyl)benzylamine, and unsubstituted benzylamine exhibited excellent agreement with values calculated from steady-state isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Hartmann
C
C
Department of Chemistry, University of California, Berkeley 94720.
Klinman
J P
JP
D006631:Amine Oxidase (Copper-Containing); D001596:Benzylamines / Q000378:metabolism; D001665:Binding Sites; D003903:Deuterium; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000378:metabolism*; D045542:PQQ Cofactor; D015363:Quinolones / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001596:Benzylamines; D015363:Quinolones; C001388:6-hydroxydopa; D004295:Dihydroxyphenylalanine; D045542:PQQ Cofactor; D003903:Deuterium; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors