Mechanism of transfer of the methyl group from (6S)- methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum

A key step in the Wood- Ljungdahl pathway of acetyl-CoA synthesis

Javier Seravalli, Shaying Zhao, Stephen W. Ragsdale

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Abstract

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood- Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 57365745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pK(a) at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate- limiting S(N)2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: k(cat) = 14.7 ± 1.7 s-1, K(m) of the CFeSP = 12 ± 4 μM, and K(m) of (6S)-CH3-H4folate = 2.0 ± 0.3 μM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.

Original languageEnglish (US)
Pages (from-to)5728-5735
Number of pages8
JournalBiochemistry
Volume38
Issue number18
DOIs
StatePublished - May 4 1999

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Corrinoids
Protein Methyltransferases
Iron-Sulfur Proteins
Clostridium
Acetyl Coenzyme A
Protonation
Wood
Amides
Kinetics
Methyltransferases
Biochemistry
Organometallics
Cobalt
Rate constants
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{ba01f59ea4ab4ea3816287e8432b81fe,
title = "Mechanism of transfer of the methyl group from (6S)- methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum: A key step in the Wood- Ljungdahl pathway of acetyl-CoA synthesis",
abstract = "The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood- Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 57365745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pK(a) at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate- limiting S(N)2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: k(cat) = 14.7 ± 1.7 s-1, K(m) of the CFeSP = 12 ± 4 μM, and K(m) of (6S)-CH3-H4folate = 2.0 ± 0.3 μM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.",
author = "Javier Seravalli and Shaying Zhao and Ragsdale, {Stephen W.}",
year = "1999",
month = "5",
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doi = "10.1021/bi982473c",
language = "English (US)",
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pages = "5728--5735",
journal = "Biochemistry",
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TY - JOUR

T1 - Mechanism of transfer of the methyl group from (6S)- methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum

T2 - A key step in the Wood- Ljungdahl pathway of acetyl-CoA synthesis

AU - Seravalli, Javier

AU - Zhao, Shaying

AU - Ragsdale, Stephen W.

PY - 1999/5/4

Y1 - 1999/5/4

N2 - The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood- Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 57365745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pK(a) at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate- limiting S(N)2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: k(cat) = 14.7 ± 1.7 s-1, K(m) of the CFeSP = 12 ± 4 μM, and K(m) of (6S)-CH3-H4folate = 2.0 ± 0.3 μM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.

AB - The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood- Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 57365745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pK(a) at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate- limiting S(N)2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: k(cat) = 14.7 ± 1.7 s-1, K(m) of the CFeSP = 12 ± 4 μM, and K(m) of (6S)-CH3-H4folate = 2.0 ± 0.3 μM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.

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