A kinetic isotope effect study and transition state analysis of the S-adenosylmethionine synthetase reaction.

Markham, George D.; Parkin, David W.; Mentch, Frank; Schramm, Vern L.

doi:10.1016/s0021-9258(18)45617-7

Cited by 66 publications

(46 citation statements)

References 17 publications

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“…Thus, some asymmetric character is present in this transition state. The E. coli methionine S -adenosyl transferase has a nearly symmetrical transition state and a reaction center 14 C KIE of 1.13 . The [5′- 3 H 2 ]ATP position reports on the hybridization state of the reaction center carbon.…”

Section: Resultsmentioning

confidence: 99%

The Transition-State Structure for Human MAT2A from Isotope Effects

Firestone

Schramm

2017

J. Am. Chem. Soc.

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Human methionine S-adenosyltransferase (MAT2A) catalyzes the formation of S-adenosylmethionine (SAM) from ATP and methionine. Synthetic lethal genetic analysis has identified MAT2A as an anticancer target in tumor cells lacking expression of 5′-methylthioadenosine phosphorylase (MTAP). Approximately 15% of human cancers are MTAP−/−. The remainder can be rendered MTAP− through MTAP inhibitors. We used kinetic isotope effect (KIE), commitment factor (Cf), and binding isotope effect (BIE) measurements combined with quantum mechanical (QM) calculations to solve the transition state structure of human MAT2A. The reaction is characterized by an advanced SN2 transition state. The bond forming from the nucleophilic methionine sulfur to the 5′-C of ATP is 2.03 Å at the transition state (bond order of 0.67). Departure of the leaving group triphosphate of ATP is well advanced and forms a 2.32 Å bond between the 5′-C of ATP and the oxygen of the triphosphate (bond order of 0.23). Interaction of MAT2A with its MAT2B regulatory subunit causes no change in the intrinsic KIEs, indicating the same transition state structure. The transition state for MAT2A is more advanced along the reaction coordinate (more product-like) than that from the near-symmetrical transition state of methionine adenosyltransferase from E. coli.

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Section: Resultsmentioning

confidence: 99%

The Transition-State Structure for Human MAT2A from Isotope Effects

Firestone

Schramm

2017

J. Am. Chem. Soc.

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“…AdoMet serves in numerous metabolic roles, acting as the methyl donor in methylation of DNA, RNA, and proteins, as the propylamine donor in polyamine synthesis, and as a noncovalent corepressor of the methionine biosynthetic regulon in Escherichia coli and Salmonella typhimurium (1,2). This laboratory has been engaged in the structural and mechanistic characterization of the E. coli metK isozyme, which is a tetramer of identical 383-residue polypeptide chains (3)(4)(5)(6)(7)(8)(9)(10). Each active site binds two divalent metal ion activators (e.g.…”

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confidence: 99%

Structural and Functional Roles of Cysteine 90 and Cysteine 240 in S-Adenosylmethionine Synthetase

Reczkowski

Markham

1995

Journal of Biological Chemistry

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Site-specific mutagenesis was performed on the structural gene for Escherichia coli S-adenosylmethionine (AdoMet) synthetase to introduce mutations at cysteines 90 and 240, residues previously implicated by chemical modification studies to be catalytically and/or structurally important. The AdoMet synthetase mutants (i.e. MetK/C90A, MetK/C90S, and MetK/C240A) retained up to approximately 10% of wild type activity, demonstrating that neither sulfhydryl is required for catalytic activity. Mutations at Cys-90 produced a mixture of noninterconverting dimeric and tetrameric proteins, suggesting a structural significance for Cys-90. Dimeric Cys-90 mutants retained approximately 1% of wild type activity, indicating a structural influence on enzyme activity. Both dimeric and tetrameric MetK/C90A had up to a approximately 70-fold increase in Km for ATP, while both dimeric and tetrameric MetK/C90S had Km values for ATP similar to the wild type enzyme, suggesting a linkage between Cys-90 and the ATP binding site. MetK/C240A was isolated solely as a tetramer and differed from wild type enzyme only in its 10-fold reduction in specific activity, suggesting that the mutation affects the rate-limiting step of the reaction, which for the wild type enzyme is the joining of ATP and L-methionine to yield AdoMet and tripolyphosphate. Remarkably all of the mutants are much more thermally stable than the wild type enzyme.

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“…MATs are an excellent model system for the study of substrate promiscuity because the chemical reactivity of the cognate physiological nucleotide substrate, ATP, is independent from the nucleobase. The C5' atom, which acts as electrophile in the MAT-catalysed reaction, belongs to the sugar moiety of the nucleotide, and is therefore distant from the nucleobase 29,30 . Moreover, SAM is not an intrinsically better methyl donor than the potential products from promiscuous reactions with non-cognate NTPs (S-guanosyl-L-methionine (SGM), S-cytidyl-L-methionine (SCM), or S-uridyl-L-methionine (SUM)), since the nucleobase does not influence the sulfonium reactivity.…”

Section: Introductionmentioning

confidence: 99%

Substrate dynamics contribute to enzymatic specificity in human and bacterial methionine adenosyltransferases

Gade

Tan

Damry

et al. 2021

Preprint

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Protein conformational change can facilitate the binding of non-cognate substrates and underlie promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here we analyse human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, non-cognate substrates bind in a stable conformation to allow catalysis. In contrast, non-cognate substrates rarely sample stable productive binding modes in eMAT owing to increased mobility of an active site loop. Different cellular concentrations of substrate likely drove the evolutionary divergence of substrate specificity in these orthologs. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated level in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the combined effects of both enzyme and substrate dynamics.

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A kinetic isotope effect study and transition state analysis of the S-adenosylmethionine synthetase reaction.

Cited by 66 publications

References 17 publications

The Transition-State Structure for Human MAT2A from Isotope Effects

The Transition-State Structure for Human MAT2A from Isotope Effects

Structural and Functional Roles of Cysteine 90 and Cysteine 240 in S-Adenosylmethionine Synthetase

Substrate dynamics contribute to enzymatic specificity in human and bacterial methionine adenosyltransferases

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