Principal Investigator :   Vidya Raghunathan

The aim of the present study is to compare the biochemical characterisitics of the two enzymes PGKB and PGKC from L. mexicana. This objective involves the following different aspects: A) Enzymological studies. Extensive characterization of the enzyme activities. B) Structural studies. The X-ray structure of various PGK’s from different sources is known. PGKC, which is different from other homologues has also been studied but lacking the c-terminal extension. It is important to know the function and role of this part of the protein.

Measurements on PGKB were continued. Data on Km and Vmax values were refined by doing the activity measurements at a wider range of substrate concentrations using both substrates of the enzyme. Yeast PGK was purified from bakers yeast using first DEAE anion exchange and then molecular sieve chromatography on a G-75 column. The conformational stability of PGKB and yeast enzyme was compared in buffered salt solutions using circular dichroism and fluorescence spectroscopy. This data were obtained in the presence of varying concentrations of denaturants and at varying temperatures. This data is being analyzed.

With PGKC the protein amount has been limiting. The expression of this protein continues to be low. The intial problem appeared to be with plasmid instability. This was overcome by recloning the pgkC gene in a different vector. The C-terminal fragment of PGKC (62 residue long) had been stably cloned in pET21c and therefore the first thing we tried was to use the same vector for PGKC. The pgk gene was removed from the pET3a and religated into pET21c. While the plasmid seemed more stable no change in the PGKC protein expression levels was observed. However the level of contaminating proteins was a little lower making the purification of PGKC somewhat easier. The main effort now is to obtain large enough quantities of the protein to have enough material for the enzymatic and spectroscopic measurements.