CATALYTIC MECHANISM OF YEAST ADENOSINE 5'-MONOPHOSPHATE DEAMINASE - ZINC CONTENT, SUBSTRATE-SPECIFICITY, PH STUDIES, AND SOLVENT ISOTOPE EFFECTS

Adenosine 5'-monophosphate (AMP) deaminase from baker's yeast is an al losteric enzyme containing a single AMP binding site and two ATP regul atory sites per polypeptide [Merkler, D. J., & Schramm, V. L. (1990) J . Biol Chem. 265, 4420-44261. The enzyme contains 0.98 +/-0 .17 zinc a tom per subunit. The X-ray crystal structure for mouse adenosine deami nase shows zinc in contact with the attacking water nucleophile using purine riboside as a transition-state inhibitor [Wilson, D. K., Rudolp h, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284]. Alignment o f the amino acid sequence for yeast AMP deaminase with that for mouse adenosine deaminase demonstrates conservation of the amino acids known from the X-ray crystal structure to bind to the zinc and to a transit ion-state analogue. On the basis of these similarities, yeast AMP deam inase is also proposed to use a Zn2+-activated water molecule to attac k C6 of AMP with the displacement of NH3. The pK(m) and pK(i) profiles for AMP and a competitive inhibitor overlap in a bell-shaped curve wi th pK(a) values of 7.0 and 7.4. This pattern is characteristic of a ra pid equilibrium between AMP and the enzyme, thus confirming the rapid equilibrium random kinetic patterns [Merkler, D. J., Wali, A. S., Tayl or, J., Schramm, V. L. (1989) J. Biol. Chem. 264, 21422-21430]. The V( max) of the reaction requires one unprotonated and one protonated grou p with pK(a) values of 6.4 +/- 0.2 and 7.7 +/- 0.3, respectively. The (H2O)-H-2-induced shifts of the pK(a) values for these groups are cons istent with a carboxylate and a histidine, groups known to be in conta ct with purine riboside in the adenosine deaminase structure. The V(ma x)/K(m) profile is similar except that the pK(a) values are 6.7 and 7. 3, respectively. Kinetic studies with (H2O)-H-2 as solvent gave invers e V(max)/K(m), solvent deuterium isotope effects, i.e., reaction rates more rapid in (H2O)-H-2 than H2O. Solvent (H2O)-H-2 isotope effects v aried from 0.79 +/- 0.11 to 0.33 +/- 0.03, with the slowest substrates giving the largest isotope effects. Proton inventory studies with a s low substrate indicated that two or more protons give rise to the solv ent isotope effect. The results are interpreted in a mechanism where e quilibrium proton transfers from the zinc-bound water and/or a compres sed hydrogen bond to the substrate contribute to the observed inverse solvent isotope effect.