Complementary Truncations of a Hydrogen Bond to Ribose Involved in Transition-State Stabilization by Cytidine Deaminase

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• 作者：Dean C. Carlow ; Steven A. Short ; and Richard Wolfenden
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：February 3, 1998
• 年：1998
• 卷：37
• 期：5
• 页码：1199 - 1203
• 全文大小：90K
• 年卷期：v.37,no.5(February 3, 1998)
• ISSN：1520-4995

文摘

The crystal structure of the complex formed betweenEscherichia coli cytidine deaminase andthe transition-state analogue inhibitor 3,4-dihydrouridine [Betts, L.,Xiang, S., Short, S. A., Wolfenden,R., & Carter, C. W. (1994) J. Mol. Biol. 235, 635] showsthe presence of an H-bond between Glu-91 andthe 3'-OH group of substituent ribose, a part of the substrate that isnot directly involved in its chemicaltransformation. To test the contribution of this interaction totransition-state stabilization, Glu-91 wasconverted to alanine. The mutant enzyme is very much less activethan the wild-type enzyme, with a500-fold increase in K_m and a 32-fold reductionin k_cat using cytidine as substrate. Nochange in secondarystructure is evident in the circular dichroic spectrum. Asmeasured by k_cat/K_m,Glu-91 thus appears tostabilize the transition state for cytidine deamination by an overallfactor of 1.7 × 10⁴, equivalent to -5.8kcal/mol in free energy. To test the contribution of thisinteraction in the opposite sense, the 3'-OHgroup of the substrate was replaced by a hydrogen atom. Comparing3'-deoxycytidine with cytidine, thenative enzyme shows a 17-fold increase in K_m anda 400-fold decrease in k_cat, indicating that the3'-hydroxyl group of cytidine stabilizes the transition state fordeamination by an overall factor of 6.3 ×10³, equivalent to -5.2 kcal/mol in free energy, asmeasured by k_cat/K_m.After one binding partner hasbeen removed, however, the effect of removing the remaining partner isrelatively slight. For the mutantenzyme E91A, removal of the 3'-hydroxyl group from substrate cytidinereduces k_cat/K_m by afactor ofonly 3. Complete removal of substituent ribose reduces thewild-type enzyme'sk_cat/K_m by a factorofmore than 10⁸; thus, substituent ribose, although distantfrom the site of chemical transformation of thesubstrate, contributes at least 11 kcal to the free energy ofstabilization of the transition state for cytidinedeamination, matching the apparent contribution to transition statebinding made by the 4-OH group ofthe pyrimidine ring, which is at the site of substrate transformation[Frick, L., Yang, C., Marquez, V. E.,& Wolfenden, R. (1989) Biochemistry 28, 9423].