Date of Award

5-2010

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

Nina M. Goodey

Committee Member

Johannes P. Schelvis

Committee Member

John J. Siekierka

Abstract

Indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (SsIGPS) belongs to a broad family of (βα)8-barrel enzymes. It catalyzes the fifth step in tryptophan biosynthesis, converting l-(o-carboxylphenylamino)-l-deoxyribulose-5-phosphate (CdRP) to indole-3-glycerol phosphate (IGP). Site selective mutagenesis was used to introduce a single cysteine in two loops near the active site, generating two recombinant proteins, each containing a single cysteine handle. The two construct were labeled, each with two different thiol-reactive probes generating four labeled constructs that were used for this study. Steady-state kinetic parameters of the labeled mutants and the wild type SsIGPS were well characterized using fluorescence spectroscopy. Subsequent experiments under single turnover (STO) conditions were employed. In the STO experiments stopped-flow instrument was used to observe IGP accumulation and change in emission of the fluorophores, to identify microscopic rate constants, and the conformational motions occurring within this enzyme. Significant changes in the fluorescence emission of the probes upon binding of IGP and substrate analog rCdRP were used to determine the binding parameters of the ligands. A mechanism was proposed for the pathway employed by SsIGPS, and was subsequently used to fit STO data for each labeled construct in a global fit using the DynaFit Program, to generate rate constants. Subsequently a mechanism for the conformational motion was proposed and the change in fluorescence intensity versus time data for the labeled constructs was fitted to this mechanism. Kinetic and conformational motion rate constants obtained from the fits were compared to fully map the entire catalytic pathway employed by SsIGPS and to determine the involvement of conformational motions in catalysis.

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