title: Kinetic mechanism and the rate-limiting step of Plasmodium vivax serine hydroxymethyltransferase.
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.
authors: From the Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand 10400, the Department of Biochemistry, Faculty of Science, Burapha University, Chonburi, Thailand 20131.
authors: Somchart
authors: S
authors: Maenpuen
authors: From the Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand 10400.
authors: Watcharee
authors: W
authors: Amornwatcharapong
authors: the Department of Biochemistry, Faculty of Science, Burapha University, Chonburi, Thailand 20131.
authors: Pasupat
authors: P
authors: Krasatong
authors: the Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 10300.
authors: Jeerus
authors: J
authors: Sucharitakul
authors: the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, and.
authors: Bruce A
authors: BA
authors: Palfey
authors: the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand 12120.
authors: Yongyuth
authors: Y
authors: Yuthavong
authors: the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand 12120.
authors: Penchit
authors: P
authors: Chitnumsub
authors: the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand 12120 ubolsree@biotec.or.th.
authors: Ubolsree
authors: U
authors: Leartsakulpanich
authors: From the Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand 10400, pimchai.cha@mahidol.ac.th.
authors: Pimchai
authors: P
authors: Chaiyen
mesh_terms: D005492:Folic Acid / Q000737:chemistry; D005998:Glycine; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010966:Plasmodium vivax / Q000201:enzymology*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D015800:Protozoan Proteins; D012694:Serine; D005492:Folic Acid; D012696:Glycine Hydroxymethyltransferase; D005998:Glycine
keywords: Amino Acid; Enzyme Kinetics; Folate; Kinetics; Pre-steady-state Kinetics; Pyridoxal Phosphate