MUTATIONS AFFECTING TRANSITION-STATE STABILIZATION BY RESIDUES COORDINATING ZINC AT THE ACTIVE-SITE OF CYTIDINE DEAMINASE

Cytidine deaminase from Escherichia coli contains 1 mol of tightly bou nd zinc per enzyme subunit (Yang, C., Carlow, D., Wolfenden, R., & Sho rt, S. A. (1992) Biochemistry 31, 4168-4174). When the metal liganding residues Cys-129 and Cys-132 were replaced by Ala, and His-102 was re placed,by Ala, Asn, or Gln, deaminase activities of cell extracts cont aining these mutant enzymes were decreased by several orders of magnit ude relative to that of the wild-type enzyme. After purification, each mutant protein was found to contain less than 0.2 mol of zinc per enz yme subunit, except mutant H102Q, which contained 1 mol of zinc per su bunit. The activity of each mutant enzyme increased in the presence of added zinc but never attained wild-type activity. Mutant H102N was un ique in that this protein could be purified as a stable apoenzyme, act ivated by added zinc, and then inhibited by EDTA. This mutant enzyme b ound zinc with an apparent K-d value of 6.0 x 10(-10) M and regained m aximal activity in the presence of 1 mol of zinc per subunit. Affiniti es of the mutant cytidine deaminases for the transition-state analogue , 5-fluoropyrimidin-2-one ribonucleoside (3,4) hydrate, were found to decrease in rough proportion to k(cat)/K-m over a range spanning sever al orders of magnitude. This variation in catalytic efficiency arose m ainly from effects on k(cat), indicating the involvement of zinc coord ination in the catalytic process rather than in substrate binding.