Site-specific isotope labeling studies of yeast phosphoglycerate kinase

Author

John Allan Lamerdin

Date of Award

12-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin Serpersu

Committee Members

Elizabeth E. Howell, Cynthia B. Peterson, Salil K. Niyogi

Abstract

Yeast phosphoglycerate kinase (PGK) is a 47 kDa glycolytic enzyme composed of 415 amino acids. By nuclear magnetic resonance (NMR) spectroscopic standards, this is a large protein, and difficult to study using conventional NMR techniques. To demonstrate the feasibility of the study of large proteins by NMR, three isotopically different forms of yeast PGK were biosynthetically prepared. Firstly, perdeuterated yeast PGK was prepared that contained isotopically normal tyrosine residues. This enzyme was characterized using one and two dimensional NMR experiments. Titration studies of this enzyme with the substrates MgATP and the competitive inhibitor glycerol-3-phosphate (G3P) indicated that at least one of the labeled tyrosine residues was located in close spatial proximity to the MgATP binding site, and whose local magnetic environment was altered upon substrate binding. Addition of G3P had minimal effect on the conformation of the enzyme. The second enzyme to be biosynthetically prepared was perdeuterated PGK Containing ¹³C_α tyrosine, ¹⁵N phenylalanine and ¹⁵N isoleucine. This enzyme was prepared in order to permit identification of two of the seven tyrosine residues found in the primary sequence of PGK. Initial characterization of this enzyme by NMR techniques was successful, however conclusive identification of the tyrosine residues was not achieved. The third enzyme to be biosynthetically prepared was perdeuterated PGK containing ¹³C_α tyrosine and ¹⁵N valine. This enzyme was also prepared to permit identification of two tyrosine residues. Using one, two and three dimensional NMR techniques, tyrosine residues 48 and 56 were conclusively identified in NMR spectra of this enzyme. This work represents the first successful attempt to conclusively identify two individual residues of a large protein using NMR methodology. It further demonstrates the feasibility structural studies of large proteins using nontraditional isotope labeling strategies in conjunction with traditional NMR techniques. Expression of yeast PGK in an auxotrophic system would greatly enhance the incorporation levels of isotopes included in the growth media of the cells. However, the availability of auxotrophic yeast strains is limited. It was decided, therefore, to express the yeast gene in a bacterial system. The yeast PGK gene was cloned and ligated into a vector containing the T7 RNA polymerase promoter site. E. coli cells of the strain BL21(DE3)pLysS were then transformed with this construct. It was observed that, although the yeast protein was expressed, the expression levels obtained were comparable to those achievable using the yeast system. The association of yeast PGK with the glycolytic enzyme glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was also investigated using small-angle neutron scattering (SANS) techniques. It has been proposed that these enzymes, which are successive in the glycolytic pathway, form a complex in the cell in order to facilitate transfer of the PGK reaction product 1,3-bisphosphoglycerate. Preliminary analysis of SANS data suggests that these enzymes do associate to allow such a transfer.

Recommended Citation

Lamerdin, John Allan, "Site-specific isotope labeling studies of yeast phosphoglycerate kinase. " PhD diss., University of Tennessee, 1997.
https://trace.tennessee.edu/utk_graddiss/9549