MUTATIONS AFFECTING TRANSITION-STATE STABILIZATION BY RESIDUES COORDINATING ZINC AT THE ACTIVE-SITE OF CYTIDINE DEAMINASE
MUTATIONS AFFECTING TRANSITION-STATE STABILIZATION BY RESIDUES COORDINATING ZINC AT THE ACTIVE-SITE OF CYTIDINE DEAMINASE
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DOI：
10.1021/bi00187a012
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发表时间：
1994-05-31
期刊：
影响因子：
2.9
通讯作者：
SHORT, SA
中科院分区：
文献类型：
--
作者：
SMITH, AA;CARLOW, DC;SHORT, SA
大肠杆菌胞苷脱氨酶每个酶亚基含有1 mol紧密结合的锌（Yang， C, Carlow， D, Wolfenden， R., & Short， S. A. (1992) Biochemistry 31, 4168-4174）。当金属配位残基Cys-129和Cys-132被Ala取代，His-102被Ala、Asn或Gln取代时，含有这些突变酶的细胞提取物的脱氨酶活性相对于野生型酶降低了几个数量级。纯化后，除突变体H102Q每个酶亚基含有1 mol锌外，每个突变蛋白的锌含量均低于0.2 mol。在添加锌的情况下，每个突变体酶的活性都有所增加，但从未达到野生型活性。突变体H102N的独特之处在于，该蛋白可以被纯化为稳定的脱酶，通过添加锌激活，然后被EDTA抑制。该突变酶结合锌的表观K-d值为6.0 x 10(-10) M，并且在每个亚基存在1 mol锌时恢复最大活性。突变胞苷脱氨酶对过渡态类似物5-氟嘧啶-2- 1核糖核苷（3,4）水合物的亲和性在几个数量级的范围内以k(cat)/ k -m的大致比例下降。这种催化效率的变化主要来自于对k（cat）的影响，表明锌配位参与了催化过程，而不是底物结合。
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 K-d 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 k(cat)/K-m over a range spanning several orders of magnitude. This variation in catalytic efficiency arose mainly from effects on k(cat), indicating the involvement of zinc coordination in the catalytic process rather than in substrate binding.