“Investigating Dipeptide Inhibition of phosphorylating Glyceraldehyde 3-phosphate dehydrogenase (GAPC)”
Glyceraldehyde 3-phosphate dehydrogenase, specifically GAPC1 (EC 126.96.36.199), is a key enzyme in the metabolic pathway of glycolysis. Previous reports have shown that inhibiting this enzyme by the dipeptide Tyr-Asp redirects the carbon flux to the pentose phosphate pathway (PPP), increasing the NADPH/NADP+ ratio and improving oxidative stress tolerance. This work aimed to investigate Tyr-Asp specificity by testing the activity of purified recombinant Arabidopsis GAPC1 against a panel of linear dipeptides. The enzymatic activity of GAPC1 was determined spectrophotometrically by following NAD+ reduction at 340 nm. In addition to the linear dipeptides, we also included a selection of cyclic dipeptides (CDPs). CDPs have amassed significant attention due to their unique and diverse chemical and biological properties, including antitumor, antifungal, antibacterial, and radical-scavenging activities. In contrast to the linear dipeptides, which are degraded inside the cell to amino acids, CDPs are highly stable. In addition to Tyr-Asp, our analysis revealed additional dipeptides and CDPs that can affect GAPC1 activity. Among the tested compounds, cyclo(His-Pro) was the strongest inhibitor with IC50 estimated to 0.31 mM. Finally, with the aim of gaining insight into the physiological effect of dipeptides and CDPs, I optimized an enzymatic assay to measure NADPH and NADP+ redox equivalents in future in vivo experiments. Progress on this project is still ongoing as there still exist open questions about the mechanism of the dipeptide inhibition of GAPC1. In the future, we hope that a full understanding of this interaction will allow for the development of more efficient means of improving plant growth under environmental stress conditions.
This past summer I had the opportunity to conduct research to improve plant growth under stress conditions. In the process, I learned an array of laboratory and biochemistry techniques such as recombinant protein expression, ion exchange chromatography with FPLC, as well as Bradford and spectrophotometric-based assays. Under the mentorship of the Skyricz lab, I was able to get a better picture of what it is like to be a researcher in plant biology. Furthermore, the opportunities offered by the Plant Genome Research Program (PGRP) through BTI during the past ten weeks such as the graduate school info panel and bioinformatics course gave me significant insights into possible career paths I might pursue in the future.