Adenosine Deaminase and Adenylate Deaminase: Comparative Kinetic Studies with Transition State and Ground State Analog Inhibitors

A number of transition state analogues and other inhibitors have been investigated by stopped-flow kinetic techniques with respect to inhibition of rabbit muscle adenylate deaminase and calf intestinal adenosine deaminase. The inhibitors are classified into three groups: (1) those which inhibit rapidly as exemplified by purine riboside or purine ribotide and which can be termed ground state analogue inhibitors, (2) those which inhibit rapidly but the initial enzyme-inhibitor complex undergoes a slow conformational change with further inhibition as exemplified by the inhibition of adenosine deaminase by 9-(erythro-2-hydroxy-3-nonyl)adenine (EHNA), and (3) those which appear to inhibit slowly as exemplified by the transition-state analogue coformycin. For this latter class, the rate constants for the on reaction have been determined as well as the overall inhibition constants. What appears to be characteristic of inhibition by different transition-state analogues is not the tightness of binding (which ranges from 5 × 10^-6 to ^10-12 M) but the apparent slow rate constant (k_on).with which inhibitor binds to the enzyme. It is concluded that the slow on rates reflect an extremely weak initial binding of the inhibitor followed by a conformational change. What appears to be characteristic of ground state analogue inhibitors is not the weakness of binding (which ranges from 2 X 10⁷ to 1.2 X 10⁵ M) but the apparent instantaneous inhibition. Thus, the initial structure of the active site of the enzyme appears to be appropriate for binding the ground state of the substrate and not the transition state, and considerable readjustment of the site seems required to bind the transition state effectively. By use of stopped-flow techniques, some estimates of the dissociation constant for the initial binding step for the transition-state analogues can be made. In the course of the work, two new transition state analogue inhibitors are reported for adenylate deaminase: coformycin 5-phosphate and 1,6- dihydro-6-hydroxymethylpurine ribotide. The chemical synthesis of the latter compound and an improved synthesis of purine ribotide are reported.