Structural analysis of catalytic and regulatory mechanisms of E. coli glutamine PRPP amidotransferase

Joseph Michael Krahn, Purdue University

Abstract

Glutamine phosphoribosylpyrophosphate amidotransferase (GPATase) catalyzes the first committed step of de novo purine biosynthesis, transfer of the amide nitrogen from glutamine to phosphoribosylpyrophosphate (PRPP), to produce phosphoribosylamine (PRA), pyrophosphate (PPi) and glutamate. GPATase is an allosteric enzyme, and the primary regulatory enzyme of purine biosynthesis. The GPATase subunit consists of 504 amino acids and is made up of two structural domains. The N-terminal domain catalyzes the abstraction of nitrogen from glutamine to produce ammonia and glutamate, and is a glutamine amidotransfer or GAT domain. This domain is a member of the Ntn amidotransferase family, and related to a diverse group of homologous enzymes termed the Ntn hydrolase family. Ntn hydrolases share a structurally and functionally similar catalytic N-terminal nucleophile in which the N-terminal amino group is believed to function as a proton donor. The C-terminal domain catalyzes the substitution of the PRPP pyrophosphate group with nitrogen obtained from the GAT domain, to produce PRA and pyrophosphate. The C-terminal domain is a member of the predominant Type I phosphoribosyltransferase (PRTase) family of enzymes. Numerous Type I PRTase crystal structures have led to the general conclusion that a “long, flexible loop” closes the active site during catalysis to provide the essential function of sequestering the activated substrate from bulk solvent. Flexible loops of all previous Type I PRTase structures have been either open or disordered, and apparently do not represent catalytically competent conformations. This work has focused on determining the mechanism of nitrogen transfer between the two catalytic sites, and on determining the catalytically active structure of the PRTase domain. Both of these key questions, required to accurately understand GPATase catalysis, have been addressed successfully by determination of the active catalytic conformation of GPATase complexed with substrate analogs in each active site. The active GPATase structure has significantly enhanced current knowledge about the general catalytic mechanism of PRTases and has suggested a model for nitrogen transfer between coupled GAT and nitrogen acceptor domains. Comparison of the active structure to an inhibitor-complexed structure has also provided significant information about the allosteric regulatory mechanism of GPATase.

Degree

Ph.D.

Advisors

Smith, Purdue University.

Subject Area

Biochemistry|Molecular biology|Microbiology

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