The Rate Constant Describing Slow-Onset Inhibition of Yeast AMP Deaminase by Coformycin Analogues Is Independent of Inhibitor Structure

(R)- and (S)-2′-deoxycoformycin, (R)-coformycin, and the corresponding 5′-monophosphates were compared as inhibitors of yeast AMP deaminase. The overall inhibition constants ranged from 4.2 mM for (S)-2′-deoxycoformycin to 10 pM for (R)-coformycin 5′-monophosphate, a difference of 3.8 × 10⁸ in affinities. (R)-Coformycin, (R)-2′-deoxycoformycin 5′-monophosphate, and (R)-coformycin 5′-mono-phosphate exhibited both rapid and slow-onset inhibition. The S inhibitors and (R)-2′-deoxycoformycin exhibited classical competitive inhibition but no time-dependent onset of inhibition. The results indicate that the presence of the 2′-hydroxyl and 5′-phosphate and the R stereochemistry at the C-8 position of the diazepine ring are necessary for the optimum interaction of inhibitors with yeast AMP deaminase. This differs from the results for rabbit muscle AMP deaminase [Frieden C., Kurz, L. C., & Gilbert, H. R. (1980) Biochemistry 19, 5303-5309] and calf intestinal adenosine deaminase [Schramm, V. L., & Baker, D. C. (1985) Biochemistry 24, 641-646], in which a tetrahedral hydroxyl at C-8 in the R stereochemistry is sufficient for slow-onset inhibition with the coformycins. The results suggest that the transition state contains a tetrahedral carbon with the R configuration as a result of the direct attack of an oxygen nucleophile at C-6 of AMP. Slow-onset inhibition of yeast AMP deaminase is consistent with the mechanism [formula omitted] in which the combination of E and I is rapidly reversible. For these inhibitors, varied by a factor of 3 × 10³, and the overall inhibition constant (K_i*) varied by a factor of 2 × 10⁵. The rate k₅, which induces slow-onset inhibition, was similar for the three slow-onset inhibitors while k₆ varied by a factor of 650, suggesting that k₅ is independent of inhibitor structure. The ability to act as a slow-onset inhibitor is a result of the ability of the inhibitor to stabilize the EI* complex. These findings argue against inhibitor-induced transition-state conformations for the slow-onset inhibitors and suggest that the enzyme attains the transition-state configuration at a low rate independent of the inhibitor structure.