MAJOR CONTRIBUTION OF A CARBOXYMETHYL GROUP TO TRANSITION-STATE STABILIZATION BY CYTIDINE DEAMINASE - MUTATION AND RESCUE

被引:49
作者
CARLOW, DC
SMITH, AA
YANG, CC
SHORT, SA
WOLFENDEN, R
机构
[1] UNIV N CAROLINA,DEPT BIOCHEM & BIOPHYS,CHAPEL HILL,NC 27599
[2] UNIV N CAROLINA,LINEBERGER COMPREHENS CANC CTR,CHAPEL HILL,NC 27599
[3] WELLCOME RES LABS,RES TRIANGLE PK,NC 27709
关键词
D O I
10.1021/bi00013a010
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The crystal structure of an inhibitory complex formed between Escherichia coli cytidine deaminase and the transition-state analog 3,4-dihydrouridine indicates the presence of a short I-I-bond between Glu-104 and the inhibitor. To test the possibility that analogous H-bonds might play a significant role in stabilizing the hydrated substrate in the transition state for deamination, we replaced Glu-104 by alanine, Compared with the wild-type enzyme, the mutant enzyme's affinities for substrate cytidine and product uridine were found to have increased, whereas k(cat) for deamination of cytidine had been reduced by 8 orders of magnitude. By its presence, the carboxymethyl group of Glu-104 appears to minimize the activation barrier for deamination, not only by stabilizing the altered substrate in the transition state but also by destabilizing the enzyme-substrate and enzyme-product complexes. In the presence of added formate ion, but not in the presence of bulkier carboxylic acids, the low catalytic activity of the mutant enzyme was enhanced substantially.
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页码:4220 / 4224
页数:5
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