The non-steady state nature of the inhibition of milk xanthine oxidase by allopurinol and alloxanthine was demonstrated, and the kinetic data presented are consistent with the known mechanisms of inhibitions by these inhibitors. With the use of human erythrocytic adenosine deaminase and its tight-binding inhibitor, coformycin, it was demonstrated that the classical methods of enzyme kinetics based on the steady state assumptions are grossly inadequate for determining the inhibition mechanisms or inhibition constants for tight-binding inhibitors. The application of the Ackermann-Potter plot, I50. the Easson-Stedman plot (or Henderson plot), and the rates of association and dissociation of enzyme-inhibititor complex were presented and their usefulness was evaluated. The molar equivalency and the catalytic number of human erythrocytic adenosine deaminase were estimated at about 1.0 × 10-10 mole/unit and 1.0 × 104 min-1 respectively. It was also demonstrated that the Ki, value of coformycin for this enzyme does not exceed 1.2 × 10-10 M, and that the second-order rate constant for the association of the enzyme with coformycin is approximately 2 × 106 M-1 sec-1. The biphasic nature of the dissociation of the deaminase-coformycin complex (EI complex) indicates that the EI complex undergoes a slow conformational change. The implications of these new kinetic approaches for the study of tight-binding inhibitors, including transition-state analogs, were discussed.
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