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

The crystal structure of an inhibitory complex formed between Escheric hia coli cytidine deaminase and the transition-state analog 3,4-dihydr ouridine indicates the presence of a short I-I-bond between Glu-104 an d the inhibitor. To test the possibility that analogous H-bonds might play a significant role in stabilizing the hydrated substrate in the t ransition state for deamination, we replaced Glu-104 by alanine, Compa red with the wild-type enzyme, the mutant enzyme's affinities for subs trate cytidine and product uridine were found to have increased, where as 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 stabi lizing the altered substrate in the transition state but also by desta bilizing the enzyme-substrate and enzyme-product complexes. In the pre sence of added formate ion, but not in the presence of bulkier carboxy lic acids, the low catalytic activity of the mutant enzyme was enhance d substantially.