Dc. Carlow et al., MAJOR CONTRIBUTION OF A CARBOXYMETHYL GROUP TO TRANSITION-STATE STABILIZATION BY CYTIDINE DEAMINASE - MUTATION AND RESCUE, Biochemistry, 34(13), 1995, pp. 4220-4224
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.