Pyridoxamine-pyruvate transaminase. I. Determination of the active site stoichiometry and the pH dependence of the dissociation constant for 5'-deoxypyridoxal

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Abstract
Spectrophotometric titration of pyridoxamine-pyruvate transaminase (EC 2.6.1.30) [from Psendomonas sp.] with pyridoxal at pH 7.15 gives 4 equivalent binding sites/tetramer. The pH dependence of the equilibrium constant for the association of 5''-deoxypyridoxal with the active site lysine residue was determined spectrophotometrically. These Kd increase with increasing pH over the range pH 7.5-9 and are correlated with the values obtained from fast reactions kinetics. In addition to this specific reaction at an active site lysine residue, a 2nd slower reaction at non-active site residues is observable at pH values greater than 8. The pH dependencies of the association and dissociation rate constants for this slow reaction were studied over the pH range 8-9 after blocking the active site by NaBH4 reduction of the pyridoxal adduct. The enzyme is stabilized and markedly activated by K+.

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