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Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: evidence for a loose transition state.

ABSTRACT: A remote labeling method has been developed to determine (18)O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of (13)C-depleted GTP and [(18)O,(13)C]GTP that contains (18)O at phosphoryl positions of mechanistic interest and (13)C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface/isotope ratio mass spectrometer. The isotope effects are 1.0012 (0.0026) in the gamma nonbridge oxygens, 1.0194 (0.0025) in the leaving group oxygens (the beta-gamma oxygen and the two beta nonbridge oxygens), and 1.0105 (0.0016) in the two beta nonbridge oxygens. The KIE in the beta-gamma bridge oxygen was computed to be 1.0116 or 1.0088 by two different methods. The significant KIE in the leaving group reveals that chemistry is largely rate-limiting whereas the KIEs in the gamma nonbridge oxygens and the leaving group indicate a loose transition state that approaches a metaphosphate. The KIE in the two beta nonbridge oxygens is roughly equal to that in the beta-gamma bridge oxygen. This indicates that, in the transition state, Ras shifts one-half of the negative charge that arises from P(gamma)-O(beta-gamma) fission from the beta-gamma bridge oxygen to the two beta nonbridge oxygens. The KIE effects, interpreted in light of structural and spectroscopic data, suggest that Ras promotes a loose transition state by stabilizing negative charge in the beta-gamma bridge and beta nonbridge oxygens of GTP.

SUBMITTER: Du X

PROVIDER: S-EPMC428437 | biostudies-literature | 2004 Jun

REPOSITORIES: biostudies-literature

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Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: evidence for a loose transition state.

Du Xinlin X Black Gavin E GE Lecchi Paolo P Abramson Fred P FP Sprang Stephen R SR

Proceedings of the National Academy of Sciences of the United States of America 20040603 24

A remote labeling method has been developed to determine (18)O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of (13)C-depleted GTP and [(18)O,(13)C]GTP that contains (18)O at phosphoryl positions of mechanistic interest and (13)C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface/isotope ratio mass spectrometer. The isotope effects are 1.0012 (0 ...[more]

PMID: 15178760

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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

Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: evidence for a loose transition state.

Publications

Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: evidence for a loose transition state.

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