Biophysical studies and characterization of enzymes in the glycolytic pathway of parasitic protozoa

Principal Investigator : Vidya Raghunathan

The aim of the present study is to compare the biochemical characteristics of the two enzymes PGKB and PGKC from L.mexicana. This objective involves the following different aspects: i) Enzymological studies; extensive characterization of the enzyme activities, ii) 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.

The earlier purification has been shortened and simplified to a single step purification method, which works for both PGKB and PGKC. This method, which has not been previously used in the case of the PGK enzymes, uses cibachron blue sepharose chromatography. Following this we perform gel permeation chromatography to give us pure enzymes.

Enzymatic characterization using NMR and biochemical methods were initiated. These are in progress. Enzyme activity is measured using a reverse assay coupling with the enzyme glyceraldehydes 3-phosphate dehydrogenase. Specific activity of the enzymes is in the range previously reported for PGK from other sources. Other kinetic properties are being investigated.

In the long-term plans discussed last year the possibility of raising polyclonal antibodies specific to PGKC, which does not cross-react with PGKB was suggested. These could be used for structure/function studies. The c-terminal extension of PGKC is the right candidate for raising such antibodies.