Structural requirements for enzymatic efficiency in cofactor-independent decarboxylation

Desai, Bijoy

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Description

Title

Structural requirements for enzymatic efficiency in cofactor-independent decarboxylation

Author(s)

Desai, Bijoy

Issue Date

2015-01-21

Director of Research (if dissertation) or Advisor (if thesis)

Gerlt, John A.

Doctoral Committee Chair(s)

Gerlt, John A.

Committee Member(s)

• Morrissey, James H.
• Nair, Satish K.
• Fratti, Rutilio A.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Date of Ingest

2015-01-21T19:49:25Z

Keyword(s)

• Enzymology
• catalysis
• Orotidine 5'-monophosphate decarboxylase
• in vitro translation
• orthogonal translation system
• non natural protein residue
• conformational change
• intrinsic binding energy
• frsA

Abstract

Orotidine 5’-monophosphate decarboxylase (OMPDC) is the last enzyme in the de novo pyrimidine biosynthetic pathway. It catalyzes the decarboxylation of orotidine 5’-monophosphate (OMP) to uridine 5’-monophosphate (UMP), without the requirement of a cofactor. It provides catalytic rate enhancements of ~1017, compared to the uncatalyzed reaction, making it one of the most efficient enzymes known. OMPDC has thus been a paradigm to understand enzymatic catalysis. Yet, many aspects of its catalytic mechanism are not well understood. The reaction coordinate consists of a vinyl carbanion/carbene intermediate. To characterize the interaction of the intermediate with the enzyme active site, I performed site-directed mutagenesis of Ser 127 to L-glycerate residue (Chapter 2). This was accomplished by in vitro protein expression using L-glycerate charged tRNA. Examination of kinetic parameters of this mutant revealed that the backbone interactions accounts for 102 fold of the total 1017 fold rate enhancement. OMPDC achieves its extraordinary rate enhancements by ground-state destabilization (GSD) and transition state destabilization (TSS). These strategies, enforced in the Michaelis complex, are accompanied by conformational changes, as characterized by the Apo (Eo) and inhibitor bound (Ec) crystal structures. A hallmark of these conformational changes is the selective formation of interactions in the Ec form compared to Eo, by residues remote from the active site of the enzyme. The role of these conformational changes in OMPDC catalysis has not been fully understood. Using a combination of site-directed mutagenesis, substrate analogs, enzyme kinetic analysis and x-ray crystallographic analysis, research in Chapter 3 attempts to elucidate the mechanism for the formation of Ec from Eo. By comparing the second order rate constants (kcat/KM) for decarboxylation of OMP and a substrate analog devoid of 5’-phosphate group (EO) for mutants of residues that interact with the 5’-phosphate group (Q185A and R203A) with those of wild-type enzyme, I have determined intrinsic binding energy (IBE) contributions of these residues. Using a similar analysis with mutants of remote residues that selectively form hydrogen bonding (T159V, R203A and Y206F) and hydrophobic (V182A) interactions in Ec, I have determined the energetic contribution of these interactions to the IBE. Combining each mutant of the “remote” interactions with that of phosphate binding interactions, I have established the energetic independence of the “remote” interactions from 5’-phosphate binding in increasing catalytic efficiency of the enzyme. Taken together, the results of this research have lead to a structural and kinetic model that explains the roles of substrate binding and conformational change in OMPDC catalysis. In Chapter 4, I investigate whether FrsA can perform cofactor independent decarboxylation of pyruvate. FrsA, a member of α/β hydrolase superfamily, was reported to catalyze cofactor independent decarboxylation of pyruvate, in a manner similar to OMPDC. Our studies show convincing evidence that frsA is not capable of catalyzing decarboxylation of pyruvate and the earlier report is incorrect.

Graduation Semester

2014-12

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Copyright 2014 Bijoy J. Desai

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