Inhibition of pyridoxal kinase by the pyridoxal-gamma-aminobutyrate imine.

Abstract

Previous studies have demonstrated that pyridoxal kinase is inhibited by y-aminobutyrate, a neurotransmitter of the vertebrate central nervous system, but the mechanism and significance of this inhibition have remained obscure.The present study was undertaken to clarify the mechanism of this inhibition.The results show that there was little inhibition of pyridoxal kinase by y-aminobutyrate at low concentrations of pyridoxal ( 4 0 0 pm), but that the inhibition became stronger as the concentration of pyridoxal was raised.Similar results were obtained when b-alanine and 8-aminovaleric acid were used as inhibitors, but glycine did not inhibit.Conventional models of enzyme inhibition did not fit the inhibition data and y-aminobutyrate did not inhibit when pyridoxamine was the substrate suggesting that it did not inhibit by interacting directly with the enzyme.Depletion of the substrate, pyridoxal, by formation of the pyridoxal-y-aminobutyrate imine and direct inhibition of pyridoxal kinase by the pyridoxal-y-aminobutyrate imine were considered as alternative mechanisms.To distinguish between the two mechanisms, the equilibrium constant for pyridoxal-y-aminobutyrate imine formation was determined and used to calculate the concentrations of free pyridoxal and pyridoxal-y-aminobutyrate imine under assay conditions.The values of K, at pH 6.2, 7.3, and 8.0 (p = 0.2 M, 37°C) were 0.20 f 0.03 M-', 3.3 f 0.3 M-', and 14.5 f 0.7 M-', respectively.The association constant for the pyridoxal-glycine imine was 0.89 2 0.12 M" (pH 6.2).The results showed that the pyridoxal concentration was not appreciably reduced by y-aminobutyrate indicating that inhibition was not the result of substrate depletion.This conclusion was supported by the failure of glycine to inhibit the enzyme.The calculated pyridoxal-y-aminobutyrate imine behaved as a simple noncompetitive inhibitor with respect to pyridoxal thereby supporting the hypothesis that pyridoxal kinase was inhibited by the imine.The relationship of the inhibition of pyridoxal kinase by pyridoxal-y-aminobutyrate imine to the synthesis of y-aminobutyrate is discussed.Pyridoxal kinase (EC 2.7.1.35),pyridoxalATP 5'-phosphotransferase, catalyzes the transfer of a phosphate group from ATP to the 5'-hydroxymethyl group of the B G vitamins (1).Pyridoxal kinase catalyzes the phosphorylation of the three forms of vitamin Be (pyridoxal, pyridoxamine, pyridoxine) at approximately equal rates.However, the Michaelis constants for the three forms vary widely.For pyridoxal kinase from