SLOW-BINDING INHIBITION OF GAMMA-AMINOBUTYRIC-ACID AMINOTRANSFERASE BY HYDRAZINE ANALOGS

(3-Hydroxybenzyl)hydrazine and methylhydrazine have been found to be p otent slow-binding inhibitors of the pyridoxal 5-phosphate (PLP)-depen dent enzyme gamma-aminobutyric acid aminotransferase (GABA-AT). Both c ompounds follow mechanism A (Morrison, J. F.; Walsh, C. T. Adv. Enzymo l. 1988, 61, 201-301) which does not involve formation of a rapidly re versible enzyme-inhibitor complex before the formation of the final ti ght complex. The rate constant for formation of the enzyme-inhibitor c omplex determined from the slow-binding kinetics was 2.08 x 10(3) and 1.98 x 10(4) M(-1) min(-1) for methylhydrazine and (3-hydroxybenzyl)hy drazine, respectively. The rate constant for dissociation of the enzym e-inhibitor complex determined for the slow-binding kinetics was 4.6 x 10(-3) and 5 x 10(-3) min(-1), respectively. The inhibition constants calculated from the slow-binding inhibition kinetics are 2.2 mu M for methylhydrazine and 0.3 mu M for (3-hydroxybenzyl)hydrazine. Reactiva tion of the inhibited enzyme was not first order, perhaps due to a sid e reaction of the hydrazine, but was consistent with the results obtai ned from the slow-binding kinetics. Inhibition constants were calculat ed from the level of enzyme activity at equilibrium inhibition. These constants are 2.8 and 0.46 mu M for methylhydrazine and (3-hydroxybenz yl)hydrazine, respectively, in good agreement with those calculated fr om the slow-binding inhibition kinetics. 3-Hydrazinopropionate also be haved as a slow-binding inhibitor. However, the dependence of its kine tics on the concentration of inhibitor could not be described by the s low-binding or slow, tight-binding inhibition models. These kinetics c ould not be described by the tight-binding character of the inhibition because the addition of the competitive inhibitor propionic acid at 1 00 times its K-i did not affect the shape of the curve for inhibitor c oncentration dependence. The slow-binding inhibition appeared to requi re 2-4 molecules of 3-hydrazinopropionate/enzyme. The reactivation of enzyme inhibited by 3-hydrazinopropionate was first order with a rate constant of 6.9 x 10(-3) min(-1). Its equilibrium inhibition constant was calculated to be <20 nM. However, the inhibition constant calculat ed was dependent on the concentration of inhibitor because of the unus ual character discussed above and may be much lower. Only 1 PLP/enzyme dimer reacted with methylhydrazine or (3-hydroxybenzyl)hydrazine, as indicated by Scatchard plots, or with 3-hydrazinopropionate, as shown by a spectrophotometric titration. Slow-binding inhibition does not ap pear to be the result of a significant enzyme conformational change be cause there is no change in the tryptophan fluorescence of GABA-AT upo n binding either methylhydrazine or 3-hydrazinopropionate. Implication s for the design of hydrazine inhibitors of GABA-AT are discussed.