Purification and characterization of fully deuterated yeast phosphoglycerate kinase

Author

Celeste Geron Shibata

Date of Award

12-1992

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin H. Serpersu

Committee Members

Elizabeth Howell, Jorge Churchich

Abstract

Fully deuterated yeast phosphoglycerate kinase was purified from yeast strain 20B-12, using plasmid pCGY219 to overproduce the enzyme. The yeast cells were grown in D₂O with deuterated algal hydrolysate as a source of amino acids and deuterated ethanol as a carbon source. The enzyme was ascertained to be fully deuterated by comparison of its one-dimensional ¹H NMR spectrum with that of a matched sample of the normal enzyme. The deuterated enzyme was then characterized by kinetic and binding studies to determine that its properties were not significantly different from those of the normal enzyme, and that the deuterated enzyme would thus be suitable for further structural studies. The deuterated enzyme was found to have K_m values of 162 µM for MgATP and 168 µM for phosphoglycerate; these are in good agreement with the K_m values of the normal enzyme. The V_maxvalue of the deuterated enzyme, 373 µmol/mg.min, is also in good agreement with that of the normal enzyme. In binding studies, all binding properties of the deuterated enzyme with regard to the catalytically relevant ternary complex are similar to those of the normal enzyme.

One- and two-dimensional NMR studies were done on the complex formed by PGK and a substrate analogue, 2'- deoxy-ATP, using both the normal and deuterated enzymes. It was expected that the deuteration of the protein would allow the substrate resonances to be more clearly visible and less ambiguous than would be the case in studies of Mg-2'-dATP bound to the normal enzyme, and this was seen to be the case. It is hoped that further experiments of a more quantitative nature aimed at deriving bound MgATP conformation from NOE constraints will be possible using the system worked out in this research.

Recommended Citation

Shibata, Celeste Geron, "Purification and characterization of fully deuterated yeast phosphoglycerate kinase. " Master's Thesis, University of Tennessee, 1992.
https://trace.tennessee.edu/utk_gradthes/12279