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Title: Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

Abstract

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.

Authors:
; ; ; ;  [1];  [2]
  1. Vanderbilt
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
OTHEROTHER U.S. GOVERNMENT
OSTI Identifier:
1041336
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochimica et Biophysica Acta. Proteins and Proteomics; Journal Volume: 1824; Journal Issue: (2) ; 02, 2012
Country of Publication:
United States
Language:
ENGLISH
Subject:
08 HYDROGEN; CRYSTAL STRUCTURE; CRYSTALLOGRAPHY; ENZYMES; GLYCINE; HYDROGEN; LIVER; METABOLISM; PROTEINS; SARCOSINE; VALINE

Citation Formats

Luka, Zigmund, Pakhomova, Svetlana, Loukachevitch, Lioudmila V, Newcomer, Marcia E, Wagner, Conrad, and LSU). Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate. United States: N. p., 2012. Web. doi:10.1016/j.bbapap.2011.10.008.
Luka, Zigmund, Pakhomova, Svetlana, Loukachevitch, Lioudmila V, Newcomer, Marcia E, Wagner, Conrad, & LSU). Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate. United States. doi:10.1016/j.bbapap.2011.10.008.
Luka, Zigmund, Pakhomova, Svetlana, Loukachevitch, Lioudmila V, Newcomer, Marcia E, Wagner, Conrad, and LSU). Wed . "Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate". United States. doi:10.1016/j.bbapap.2011.10.008.
@article{osti_1041336,
title = {Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate},
author = {Luka, Zigmund and Pakhomova, Svetlana and Loukachevitch, Lioudmila V and Newcomer, Marcia E and Wagner, Conrad and LSU)},
abstractNote = {Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.},
doi = {10.1016/j.bbapap.2011.10.008},
journal = {Biochimica et Biophysica Acta. Proteins and Proteomics},
number = (2) ; 02, 2012,
volume = 1824,
place = {United States},
year = {Wed Jun 27 00:00:00 EDT 2012},
month = {Wed Jun 27 00:00:00 EDT 2012}
}
  • The effects of media vitamin B/sub 12/(CNB/sub 12/), L-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH/sub 4/folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mM methionine elevates the amount of B/sub 12/ methyltransferase 1.8--2.3-fold at media folate concentrations of 0.2--2.0 ..mu..M. Conversely, excess 100 ..mu..M folic acid increases the amount of B/sub 12/ holoenzyme by 2.4--3.0-fold when the medium contains 0.01--0.1 mM methionine. These increases in B/sub 12/ methyltransferase promoted by 100 ..mu..M media folate and 10mM methionine are inhibited by cycloheximide. 5-MeH/sub 4/folate will support growth and induce methyltransferase synthesismore » more efficiently than folic acid. Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 ..mu..M CNB/sub 12/, 0.1 mM homocysteine, and 100 ..mu..m folic acid or 10 ..mu..m dl-5-MeH/sub 4/folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB/sub 12/ does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mM methionine-containing to methionine-free media is dependent upon the B/sub 12/ holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 ..mu..m CNB/sub 12/ to stabilize and convert apo- to holomethyltransferase, and 100 ..mu..M folate (or 10 ..mu..M dl-5-MeH/sub 4/folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20-25 percent increase in the cellular B/sub 12/ enzyme content over the level already induced by 100 ..mu..M folate in 0.1 mM methionine-supplemented media.« less
  • Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M/sup -1/ min /sup -1/ is obtained at pH 8.2 and 25/sup 0/C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constantmore » for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of (/sup 14/C) phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.« less
  • Rat liver glycine methyltransferase is completely inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Treatment of the inactivated enzyme with KCN results in a reactivated enzyme having values of Vmax and S0.5 for S-adenosyl-L-methionine comparable to those of the native enzyme and about a 4-fold greater Km value for glycine. Kinetics of inactivation and reactivation show that one cysteine residue is involved in this process. Reaction of the methyltransferase with iodoacetate leads to partial inactivation of the enzyme; about 22% of the initial activity is retained in the modified enzyme. The relationship between the loss of enzyme activity and the number of iodoacetatemore » molecules incorporated and the sequence analysis of peptides containing the modified residues indicate that carboxymethylation of Cys-282 is responsible for loss of activity. The observations that the activity of the cyanylated glycine methyltransferase shows no decrease upon incubation with iodoacetate and, conversely, the residual activity associated with the iodoacetate-modified enzyme is not abolished by DTNB suggest that Cys-282 is also involved in the inactivation by DTNB. Besides this residue, Cys-185, Cys-246, and Cys-262 are modified upon prolonged incubation with iodoacetate. 5'-(p-(Fluorosulfonyl)benzoyl)adenosine (FSBA) inactivates glycine methyltransferase by forming 1 disulfide/subunit. Despite this stoichiometry, treatment of the FSBA-inactivated enzyme with unlabeled iodoacetate and then with iodo(/sup 14/C)acetate after reduction with 2-mercaptoethanol and subsequent peptide analysis show that the incorporated radioactivity is distributed equally among Cys-185, Cys-246, Cys-262, and Cys-282.« less