Kinetic Mechanism and the Rate-Limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase [Enzymology]

February 12th, 2015 by Maenpuen, S., Amornwatcharapong, W., Krasatong, P., Sucharitakul, J., Palfey, B. A., Yuthavong, Y., Chitnumsub, P., Leartsakulpanich, U., Chaiyen, P.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from L-serine to tetrahydrofolate (H4folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH2-H4folate). SHMT is crucial for dTMP biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary-complex mechanism. In order to define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either L-serine or H4folate. The dissociation constants for the enzyme:L-serine and enzyme:H4folate complexes were determined as 0.18 +/- 0.08 mM and 0.35 +/- 0.06 mM, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the kcat value (1.09 +/- 0.05 s-1) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development.