Role of Enzyme-Ribofuranosyl Contacts in the Ground State and Transition State for Orotidine 5'-Phosphate Decarboxylase: A Role for Substrate Destabilization?

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• 作者：Brian G. Miller ; Glenn L. Butterfoss ; Steven A. Short ; and Richard Wolfenden
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：May 29, 2001
• 年：2001
• 卷：40
• 期：21
• 页码：6227 - 6232
• 全文大小：237K
• 年卷期：v.40,no.21(May 29, 2001)
• ISSN：1520-4995

文摘

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexedwith the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of stronginteractions between enzyme residues and functional groups of this ligand. Enzyme contacts with thephosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reactioncatalyzed by yeast ODCase. In addition to these enzyme-ligand contacts, active site residues contributedby both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OHgroups of the ligand's ribosyl moiety. These involve Thr-100 of one subunit and Asp-37 of the oppositesubunit, respectively. To evaluate the contributions of these ribofuranosyl contacts to ground state andtransition state stabilization, Thr-100 and Asp-37 were each mutated to alanine. Elimination of the enzyme'scapacity to contact individual ribosyl OH groups reduced the k_cat/K_m value of the T100A enzyme by60-fold and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMPdecreased the binding affinity by less than a factor of 10, but decreased k_cat by more that 2 orders ofmagnitude. Upon removal of the complementary hydroxymethyl group from the enzyme, little furtherreduction in k_cat/K_m for 2'-deoxyOMP was observed. To assess the contribution made by contacts involvingboth ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMPwas measured. The value of k_cat/K_m for this enzyme-substrate pair was 170 M^-1 s^-1, representing adecrease of more than 7.6 kcal/mol of binding free energy in the transition state. To the extent thatelectrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lendobvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complexplays a major role in catalysis.