Dc. Carlow et al., COMPLEMENTARY TRUNCATIONS OF A HYDROGEN-BOND TO RIBOSE INVOLVED IN TRANSITION-STATE STABILIZATION BY CYTIDINE DEAMINASE, Biochemistry, 37(5), 1998, pp. 1199-1203
The crystal structure of the complex formed between Escherichia coli c
ytidine deaminase and the transition-state analogue inhibitor 3,4-dihy
drouridine [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., & Carte
r, C. W. (1994) J. Mol. Biol. 235, 635] shows the presence of an H-bon
d between Glu-91 and the 3'-OH group of substituent ribose, a part bf
the substrate that is not directly involved in its chemical transforma
tion. To test the contribution of this interaction to transition-state
stabilization, Glu-91 was converted to alanine. The mutant enzyme is
very much less active than the wild-type enzyme, with a 500-fold incre
ase in K-m and a 32-fold reduction in k(cat) using cytidine as substra
te. No change in secondary structure is evident in the circular dichro
ic spectrum. As measured by k(cat)/K-m, Glu-91 thus appears to stabili
ze the transition state for cytidine deamination by an overall factor
of 1.7 x 10(4), equivalent to -5.8 kcal/mol in free energy. To test th
e contribution of this interaction in the opposite sense, the 3'-OH gr
oup of the substrate was replaced by a hydrogen atom. Comparing 3'-deo
xycytidine with cytidine, the native enzyme shows a 17-fold increase i
n K-m and a 400-fold decrease in k(cat), indicating that the 3'-hydrox
yl group of cytidine stabilizes the transition state for deamination b
y an overall factor of 6.3 x 10(3), equivalent to -5.2 kcal/mol in fre
e energy, as measured by k(cat)/K-m. After one binding partner has bee
n removed, however, the effect of removing the remaining partner is re
latively slight. For the mutant enzyme E91A, removal of the 3'-hydroxy
l group from substrate cytidine reduces k(cat)/K-m by a factor of only
3. Complete removal of substituent ribose reduces the wild-type enzym
e's k(cat)/K-m by a factor of more than 10(8); thus, substituent ribos
e, although distant from the site of chemical transformation of the su
bstrate, contributes at least 11 kcal to the free energy of stabilizat
ion of the transition state for cytidine deamination, matching the app
arent contribution to transition state binding made by the 4-OH group
of the pyrimidine ring, which is at the site of substrate transformati
on [Frick, L., Yang, C., Marquez, V. E., & Wolfenden, R. (1989) Bioche
mistry 28, 9423].