COMPLEMENTARY TRUNCATIONS OF A HYDROGEN-BOND TO RIBOSE INVOLVED IN TRANSITION-STATE STABILIZATION BY CYTIDINE DEAMINASE

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].