Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Somchart Maenpuen,Watcharee Amornwatcharapong,Pasupat Krasatong,Jeerus Sucharitakul,Bruce A Palfey,Yongyuth Yuthavong,Penchit Chitnumsub,Ubolsree Leartsakulpanich,Pimchai Chaiyen

doi:10.1074/jbc.m114.612275

Abstract

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 deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. 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 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.

Highlights

Plasmodium vivax serine hydroxymethyltransferase (PvSHMT) catalyzes formation of glycine from L-serine and tetrahydrofolate
Because the conditions employed only allow for a single turnover of the reactions to proceed, these data indicated that all mixing setups resulted in a similar amount of product after the glycine formation step, suggesting that all mixing setups led to a productive pathway and the PvSHMT reaction occurs via a random-order mechanism (Fig. 8B)
Our investigation based on transient kinetics, thermodynamics, and steady-state kinetics provides insight into the reaction mechanism of PvSHMT and has identified glycine formation as the rate-limiting step of this enzyme reaction

Summary

Background

Plasmodium vivax serine hydroxymethyltransferase (PvSHMT) catalyzes formation of glycine from L-serine and tetrahydrofolate. 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. Double-reciprocal plots of bi-substrate kinetics of SHMT from rabbit liver cytosol, P. vivax, and P. falciparum display intersecting lines, suggesting that the reaction occurs via a ternary complex mechanism in which the formation of an enzyme1⁄7serine1⁄7H4folate complex is required for catalysis [5, 12, 17] It was unclear whether the substrates bind in a compulsory or random order.

EXPERIMENTAL PROCEDURES

RESULTS

18 Ϯ 3 21 Ϯ 3 22 Ϯ 4 22 Ϯ 2 12 Ϯ 3 22 Ϯ 8

DISCUSSION