Aa. Smith et al., MUTATIONS AFFECTING TRANSITION-STATE STABILIZATION BY RESIDUES COORDINATING ZINC AT THE ACTIVE-SITE OF CYTIDINE DEAMINASE, Biochemistry, 33(21), 1994, pp. 6468-6474
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.