Date of Award
Doctor of Philosophy
Electrospray ionization mass spectrometry,(ESI-MS)-based assays have been newly developed to study three classes of enzymes (sugar nucleotidyltransferases, glycosyltransferases, and phosphorylases) from the three distinct families of life: archaea, eukaryotes, and prokaryotes. These three classes of enzymes play vital roles in carbohydrate storage, degradation and synthesis. The sugar nucleotidyltransferases from Escherichia coli and yeast that biosynthesize UDP-glucose and ADP-glucose respectively were studied using the first ESI-MS assay. Nonlinear regression of the kinetic data was comparable to those determined from the traditional UDP-glucose dehydrogenase coupled assay. This technique allowed us to determine that the yeast sugar nucleotidyltransferase has one binding site that biosynthesizes both ADP-glucose and UDP-glucose. A related assay was applied to the study of glycogen synthases from rabbit, yeast, and the first archaeal glycogen synthase from P. furiosus. Unlike the specificity seen in the rabbit and yeast enzymes, the P. furiosus glycogen synthase did not differentiate substrates on the basis of the nucleotide but on the structure of the carbohydrate. The P. furiosus glycogen synthase showed broad acceptance of ADP-, dTDP-, GDP-, and UDP-glucose while not accepting UDP-galactose. Competition binding experiments demonstrated that for both the rabbit muscle and P. furiosus glycogen synthases the alpha-phosphate is the most important feature for binding between the substrate and biocatalyst. Through ESI-MS analysis, the kinetic parameters KM, Vmax, and Ki (for alpha/beta-D-glucose) also were determined for an enzyme responsible for degrading glycogen polymers. The rabbit muscle phosphorylase b accepted maltoheptaose, maltohexaose, and maltopentaose but not maltotetrose as substrates in the direction of glycogen phosphorylysis. Many biocatalysts, in particular sugar nucleotidyltransferases, depend on metals for full catalytic activity; however the role they play is not well understood. Using isothermal titration calorimetry (ITC) we have studied the binding relationship between the divalent metals, magnesium and manganese, and uridine-5'-phosphates to determine the role these metals play in carbohydrate biosynthesis. The binding constant associated with the coordination of Mg+2 and Mn+2 to the phosphate was shown to follow the pattern of PPi>UTP>UDP>UMP≈UDP-Glc≈Glc-1-P. Through the use of ESI-MS and ITC we have begun to explore the binding relationship between the substrate and the biocatalyst.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Corbin James Zea
Zea, Corbin James, "Probing carbohydrate biocatalysis through the use of electrospray ionization mass spectrometry and isothermal titration calorimetry " (2005). Retrospective Theses and Dissertations. 1929.