Chemical and structural dynamics of guanosine monophosphate synthetase
Abstract
Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine to create ammonia. The enzyme family provides numerous examples for study of the regulation and interdomain communication in proteins with multiple catalytic activities. Guanosine monophosphate synthetase (GMPS) is a glutamine amidotransferase responsible for the last step in the guanosine branch of de novo purine biosynthesis, the amination of xanthosine monophosphate. In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS provided no details of the transfer process. Large-scale conformational changes in GMPS are necessary to allow efficient ammonia channeling to occur, and they are hypothesized to consist of global domain motions as well as the ordering of a long, conserved loop. Rapid kinetics studies were pursued to provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Absorbance changes that occur upon mixing of GMPS with nucleotide substrates were established to be indicative of the formation of a reactive nucleotide intermediate. Subsequent mixing with glutamine was used to directly monitor the process of ammonia transfer to this intermediate. Nucleotide-induced changes in intrinsic tryptophan fluorescence provided the overall rate for the conformational changes upon substrate binding. A kinetic model for a single turnover event has been developed that highlights distinct chemical and conformational steps in the overall process. Structural information derived from both limited proteolysis and sedimentation velocity experiments support the hypothesis of nucleotide-induced loop- and domain-closure in the protein. These results were combined with information from sequence conservation and precedents from other glutamine amidotransferases to develop a structural model of GMPS in the closed, active state. The overall results provide a more complete kinetic and structural picture of GMPS function, from the binding of nucleotide substrates to the open enzyme, the ensuing chemical and conformational changes, and the final return to an open state with release of products. Finally, comparative analysis of the GMPS from different sources has further developed the distinctions in functional roles for dimeric and monomeric versions of protein.
Degree
Ph.D.
Advisors
Davisson, Purdue University.
Subject Area
Biochemistry
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