Synergistic inhibition of xanthine oxidase by guanidinium plus thiocyanate

Abstract

Milk xanthine oxidase was inhibited competitively by guanidinium and noncompetitively by thiocyanate. When present simultaneously, these inhibitors exhibited a striking synergism manifested by an apparent mutual lowering of inhibitory constants ( K). Thus, K G + was decreasedfrom 7 × 10 −3 M to 8 × 10 −4 M by the presence of excess thiocyanate, and K S − was decreased from 0.3 to 0.01 M by the presence of excess guanidinium. It was possible to calculate inhibitory constants for each of these inhibitors in terms of their inhibition of the enzyme when acting alone and also in terms of their potentiation of the inhibition caused by the other inhibitor, when acting together. The value of K G + arrived at in these two ways was identical, whereas the value of K S − derived from its effects on guanidinium inhibition was smaller by a factor of 25 than was K S − derived from its inhibition of the enzyme when acting alone. This suggested that guanidinium binds to the enzyme at the same site, in competition with xanthine, whether thiocyanate is present or not. Thiocyanate, in contrast, binds to a noncompetitive site when acting alone and, in the presence of guanidinium, also to a site newly created for it by the binding of guanidinium. This interpretation was supported by the effects of pH on K G + , K S − , and the degree of synergism and by the observation that the presence of guanidinium converts thiocyanate to a largely competitive inhibitor.