Dataset Information

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State.

ABSTRACT: The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates.

SUBMITTER: Behiry EM

PROVIDER: S-EPMC5861672 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State.

Behiry Enas M EM Ruiz-Pernia J Javier JJ Luk Louis L Tuñón Iñaki I Moliner Vicent V Allemann Rudolf K RK

Angewandte Chemie (International ed. in English) 20180219 12

The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unit ...[more]

PMID: 29341402

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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

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State.

Publications

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State.

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