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

Project Associates / Assistants
Sangita Aggarwal

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 two aspects viz.(i) Enzymological studies. Extensive characterization of the enzyme activities. This will help us confirm thereby biological role in the cell and also compare the two enzymes functionally and (ii) Structural studies. The 3-dimensional structure of PGK’s from different sources such as yeast, pig, mouse, human etc. is known. However PGK in each of this is a cytoplasmic enzyme. Tissue specific isoforms are found in the human enzyme but they are closely similar in sequence and identical is location. Trypanosomatidae on the other hand has different types of PGK each differentially located within the cell. As the glycolysis in these organisms has many unique features, it has been suggested that the different isoforms may have a role to play in this. Since PGKC from trypanosomatidae is a vesicular form of the enzyme, it is expected to have differences from other eukaryotic homologues. Indeed PGKC has a significant sequence difference from trypanosomatidae PGKB which is the cytoplasmic form of the enzyme. Most of the differences between PGKC and PGKB lie at the C-terminus of the protein. The PGKC C-terminus is highly variable even between closely related trypanosomatids such as Trypanosoma and Leishmania. The presence of various forms of PGK within a single cell of the trypanosomatidae is thought to have a role in the stage-specific regulation and control of glycolysis although no experimental data is yet available to support the claim. It is thus our goal to understand the differences between PGKB and PGKC and determine if indeed they have an important regulatory role within the cell.

Enzyme activities of PGKB were measured at a range of substrate concentrations and K_m and V_max determined. For PGKB, complete binding data for ATP, ADP and 3-PG were thus obtained. Kinetic parameters for ADP binding to PGKB were not measured in the standard GAPDH coupled assay. A modified assay protocol was developed using pyruvate kinase/ PEP for ATP generation. The assay was standardized for optimal substrate concentrations and then used to determine K_m and V_max for ADP binding to PGKB. For PGKC K_m and V_max data have been obtained with ATP and 3-PG but not ADP.

The C-terminal sequence of PGKC was analyzed for possible functional roles by homology search in BLAST followed by sequence alignments as well as by structure prediction algorithms.