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Kinetic mechanism and the rate-limiting step of Plasmodium vivax serine hydroxymethyltransferase.
J Biol Chem. 2015 Mar 27; 290(13):8656-65.JB

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+Show Affiliations

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.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.the Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 10300.the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, and.the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand 12120.the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand 12120.the National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand 12120 ubolsree@biotec.or.th.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.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25678710

Citation

Maenpuen, Somchart, et al. "Kinetic Mechanism and the Rate-limiting Step of Plasmodium Vivax Serine Hydroxymethyltransferase." The Journal of Biological Chemistry, vol. 290, no. 13, 2015, pp. 8656-65.
Maenpuen S, Amornwatcharapong W, Krasatong P, et al. Kinetic mechanism and the rate-limiting step of Plasmodium vivax serine hydroxymethyltransferase. J Biol Chem. 2015;290(13):8656-65.
Maenpuen, S., Amornwatcharapong, W., Krasatong, P., Sucharitakul, J., Palfey, B. A., Yuthavong, Y., Chitnumsub, P., Leartsakulpanich, U., & Chaiyen, P. (2015). Kinetic mechanism and the rate-limiting step of Plasmodium vivax serine hydroxymethyltransferase. The Journal of Biological Chemistry, 290(13), 8656-65. https://doi.org/10.1074/jbc.M114.612275
Maenpuen S, et al. Kinetic Mechanism and the Rate-limiting Step of Plasmodium Vivax Serine Hydroxymethyltransferase. J Biol Chem. 2015 Mar 27;290(13):8656-65. PubMed PMID: 25678710.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic mechanism and the rate-limiting step of Plasmodium vivax serine hydroxymethyltransferase. AU - Maenpuen,Somchart, AU - Amornwatcharapong,Watcharee, AU - Krasatong,Pasupat, AU - Sucharitakul,Jeerus, AU - Palfey,Bruce A, AU - Yuthavong,Yongyuth, AU - Chitnumsub,Penchit, AU - Leartsakulpanich,Ubolsree, AU - Chaiyen,Pimchai, Y1 - 2015/02/12/ PY - 2015/2/14/entrez PY - 2015/2/14/pubmed PY - 2015/6/3/medline KW - Amino Acid KW - Enzyme Kinetics KW - Folate KW - Kinetics KW - Pre-steady-state Kinetics KW - Pyridoxal Phosphate SP - 8656 EP - 65 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 290 IS - 13 N2 - 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. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/25678710/Kinetic_mechanism_and_the_rate_limiting_step_of_Plasmodium_vivax_serine_hydroxymethyltransferase_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=25678710 DB - PRIME DP - Unbound Medicine ER -