Doctoral Dissertations

Jay Gregory

Date of Award

8-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin H. Sepersu

Committee Members

Dan Roberts, Jorge Churchich, Solon Georghiou

Abstract

The active site geometry of yeast phosphoglycerate kinase (PGK) was investigated using various nuclear magnetic resonance methods. β,γ-bidentate CrATP, a stable, exchange inert metal-nucleotide analog, was used as a probe of the conformation of 3-phospho-D-glycerate (3PGA) at the active site of PGK. The paramagnetic effects of Cr^"^ on the longitudinal relaxation rates (1/T,p) of the ^H, '^P and "c nuclei of 3PGA were examined in order to determine the distances between enzyme-bound CrATP and the nuclei of 3PGA in the ternary PGK•CrATP•3PGA complex. Kinetic studies indicated That KiCrATP was dependent on the concentration of 3PGA, and the dependence was abolished in the presence of activator sulfate ion. Similarly, NMR studies showed that the environment of CrATP bound at the active site was altered by the presence of sulfate ion.

The metal nucleus distances determined from paramagnetic NMR studies show the close proximity of the substrates at the active site of PGK, which is consistent with a hinge-bending conformational change of the enzyme for bringing the two substrates together. Additionally, the presence of an activating concentration of sulfate was observed to cause significant changes in both the relative Cr3+-3PGA distances and the overall conformation of 3PGA when compared to the findings in the absence of sulfate. This is consistent with the active site being in a "less closed" configuration when sulfate is present. Under both conditions, models can be constructed which allow for direct transfer of a phosphoryl group between the bound substrates.

Two-dimensional studies of the nuclear Overhauser effect (TRNOESY) between the protons of 2'deoxy-ATP (dATP) were used to delineate the ribose conformation and the glycosidic angle of the nucleotide bound in a ternary PGK•MgdATP•glycerol-3 phosphate complex. The resulting structure indicated an 04' endo conformation for the ribose ring and a glycosidic angle of 59°. Additionally, the dependence of the observed NOE intensities and the corresponding calculated distances was analyzed as a function of the mixing time for NOE development and of the [substrate]/[protein] ratio. It was observed that no single mixing time was adequate for reliable estimation of initial cross-relaxation rates for all interproton NOE's observed, and that although spin diffusion can be effectively circumvented for longer periods at higher [substrate]/[protein] ratios, the lack of NOE constraints under these conditions make distance calculations unreliable. The results suggest that an in-depth analysis of this type should be performed for NOE conformational studies in order to obtain the best possible structure. The results suggest that the use of a perdeuterated enzyme system as described in this study is essential for accurate estimation of cross-relaxation rates at low [substrate]/[protein] ratios.

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