Dataset Information

The Transition-State Structure for Human MAT2A from Isotope Effects.

ABSTRACT: 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.

SUBMITTER: Firestone RS

PROVIDER: S-EPMC5674783 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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The Transition-State Structure for Human MAT2A from Isotope Effects.

Firestone Ross S RS Schramm Vern L VL

Journal of the American Chemical Society 20170920 39

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 bindi ...[more]

PMID: 28880543

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Project description:Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13C KIE of 1.04, an inverse intrinsic ?-secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.

Dataset Information

The Transition-State Structure for Human MAT2A from Isotope Effects.

Publications

The Transition-State Structure for Human MAT2A from Isotope Effects.

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