Threonine deaminase: A structure-function study

Cindy Lee Wolfe, Purdue University

Abstract

Threonine deaminase is a tetramer of identical polypeptide chains with a total molecular weight of 204,000. This highly regulated enzyme catalyzes the first step in the isoleucine biosynthetic pathway. Threonine deaminase with the aid of its coenzyme pyridoxal phosphate catalyzes the deamination of its substrate threonine to $\alpha$-ketobutyrate. The goal of this project was three fold: to determine the number and location of the active site, activator site, and inhibitor site in Escherichia coli K-12 threonine deaminase; to study the regulation of an allosteric mechanism in threonine deaminase; and to relate enzyme structure to function. The wild-type ilvA gene which encodes threonine deaminase was cloned into the T7 expression vector and overexpressed such that threonine deaminase comprised 10% of total cell protein. Threonine deaminase was then purified to homogeneity. The number of active sites and effector binding sites were determined by kinetic and ligand binding studies. It was found that there are four active sites per tetramer with Lys$\sp{61}$ forming a Schiff's base linkage with pyridoxal phosphate. Substrate binding was slightly cooperative with cooperatively increasing with increasing inhibitor concentration. Tetrameric threonine deaminase was found to have four inhibitor sites which bind isoleucine with high cooperativity. Leucine and valine also inhibited the enzyme but with a much reduced affinity for the inhibitor site. Only one molecular of activator, valine, per tetramer was required to reverse isoleucine or leucine inhibition. The behavior of threonine deaminase could be reasonably well explained by the assumptions and equations of the Monod-Wyman-Changeux model. Isoleucine insensitive mutants were isolated by the ability of Escherichia coli to grow on minimal media containing the isoleucine analogue, isoleucine tetrazole. Isoleucine tetrazole is able to bind to the inhibitor site of threonine deaminase thus inhibiting isoleucine biosynthesis. The mutant ilvA gene was amplified via the polymerase chain reaction (PCR) and placed into an expression vector. Mutation threonine deaminases were purified and kinetically characterized. The mutant IlvA gene was sequenced. Consequently, specific regions of the enzyme important for allosteric regulation and ligand binding were located.

Degree

Ph.D.

Advisors

Umbarger, Purdue University.

Subject Area

Biochemistry|Molecular biology

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