title: Mutations affecting transition-state stabilization by residues coordinating zinc at the active site of cytidine deaminase.
abstract: Cytidine deaminase from Escherichia coli contains 1 mol of tightly bound zinc per enzyme subunit (Yang, C., Carlow, D., Wolfenden, R., & Short, 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 replaced by Ala, Asn, or Gln, deaminase activities of cell extracts containing these mutant enzymes were decreased by several orders of magnitude 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 enzyme subunit, except mutant H102Q, which contained 1 mol of zinc per subunit. The activity of each mutant enzyme increased in the presence of added zinc but never attained wild-type activity. Mutant H102N was unique in that this protein could be purified as a stable apoenzyme, activated by added zinc, and then inhibited by EDTA. This mutant enzyme bound zinc with an apparent Kd value of 6.0 x 10(-10) M and regained maximal activity in the presence of 1 mol of zinc per subunit. Affinities 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 kcat/Km over a range spanning several orders of magnitude. This variation in catalytic efficiency arose mainly from effects on kcat, indicating the involvement of zinc coordination in the catalytic process rather than in substrate binding.
authors.affiliation: Wellcome Research Laboratory, Research Triangle Park, North Carolina 27709.
authors.forename: A A
authors.initials: AA
authors.lastname: Smith
authors.forename: D C
authors.initials: DC
authors.lastname: Carlow
authors.forename: R
authors.initials: R
authors.lastname: Wolfenden
authors.forename: S A
authors.initials: SA
authors.lastname: Short
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D009154:Mutation; D015032:Zinc / Q000737:chemistry*
chemical_list: D003564:Cytidine Deaminase; D015032:Zinc