SUBSTRATE CONNECTIVITY EFFECTS IN THE TRANSITION-STATE FOR CYTIDINE DEAMINASE

The binding properties of substrates and competitive inhibitors of Esc herichia coli cytidine deaminase are compared with those of the fragme nts obtained by cutting these ligands at several positions including t he glycosidic bond. In contrast with the normal substrate cytidine (k( cat)/K-m = 2.6 x 10(6) M-1 s(-1)), cytosine is found to serve as an ex tremely slow substrate (k(cat)/K-m = 1.8 x 10(-3) M-1 s(-1)), despite the ability of cytosine to enter any active site that can accommodate the normal substrate cytidine. Spontaneous nonenzymatic deamination pr oceeds at similar rates for cytosine and cytidine at pH 7 and 25 degre es C, indicating that substituent ribose exerts little effect on the i ntrinsic reactivity of cytidine in solution. Dividing k(non) by k(cat) /K-m, the maximal K-d value of the enzyme's complex with the altered s ubstrate in the transition state is estimated as 6.1 x 10(-8) M for cy tosine, very much higher than the value (1.2 x 10(-16) M) estimated fo r cytidine. The K-d value of ribofuranose, the missing substituent, is roughly 1.8 x 10(-2) M, as indicated by the K-i values of D-ribose an d 1-methyl-D-ribofuranoside as competitive inhibitors. Thus, the free energy of binding of the altered substrate in the transition state is 9.5 kcal/mol more favorable for the whole molecule cytidine than for t he sum of those of its parts, cytosine plus ribofuranose. As a separat e molecule, however, ribose shows no detectable effect on the enzyme's activity on cytosine. Connectivity effects of similar magnitude are i ndicated by the equilibrium binding affinities of inhibitors. Thus, th e K-i value of the transition state analogue inhibitor zebularine hydr ate (1.2 x 10(-12) M) is very much lower than the combined affinities of N-ribofuranosylurea (1.6 x 10(-4) M) and allyl alcohol (0.14 M), in dicating that the glycoside bond, by its presence, exerts a connectivi ty effect of 9.9 kcal/mol on the observed free energy of binding.