Deuterium kinetic isotope effects in heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96: the anionic form of the substrate in the enzyme–substrate complex is a reactive species

2012 ◽  
Vol 151 (6) ◽  
pp. 633-642
Author(s):  
Mutsumi Saito ◽  
Ai Itoh ◽  
Haruo Suzuki
Keyword(s):  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Reactive Species ◽  
Anionic Form ◽  
Sarcosine Oxidase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Kinetic Isotope ◽  
Corynebacterium Sp

Zum Mechanismus der photochemischen Dehydrocyclisierung von Diphenyläther / On the Mechanism of the Photochemical Dehydrocyclisation of Diphenylether

1977 ◽  
Vol 32 (3) ◽  
pp. 285-288 ◽  
Author(s):  
Klaus-Peter Zeller ◽  
Günther Gauglitz
Keyword(s):  
Triplet State ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Reaction Scheme ◽  
Reactive Species ◽  
Kinetic Isotope

The photochemical cyclisation of diphenylether to dibenzofuran is scavenged by trans-1,3-pentadiene indicating that the triplet state of the ether is the reactive species. The intramolecular, kinetic isotope effects kʜ/kᴅ for the cyclisation of 2-deuteriodiphenyl ether and 1-phenoxy-4-pentadeuteriophenoxy-benzene are 1.12 ± 0.05 and 1.19 ± 0.05, respectively. A reaction scheme consisting of a photochemical cyclisation to 1 a, 9 a-dihydrodi-benzofuran followed by dehydrogenation to dibenzofuran is suggested.

scholarly journals Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

2016 ◽  
Vol 113 (52) ◽  
pp. E8369-E8378 ◽  
Author(s):  
Joshua A. Linscott ◽  
Kanishk Kapilashrami ◽  
Zhen Wang ◽  
Chamara Senevirathne ◽  
Ian R. Bothwell ◽  
...  
Keyword(s):  
Transition State ◽  
Cell Cycle Progression ◽  
Cancer Metastasis ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Substrate Complex ◽  
Flight Mass Spectrometry ◽  
Kinetic Isotope ◽  
Steady State Kinetics ◽  
Lysine Methyltransferase

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 cofactorS-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic13C KIE of 1.04, an inverse intrinsic α-secondary CD3KIE of 0.90, and a small but statistically significant inverse CD3BIE 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 analogSe-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.

Protein dynamics and enzyme catalysis: the ghost in the machine?

2012 ◽  
Vol 40 (3) ◽  
pp. 515-521 ◽  
Author(s):  
David R. Glowacki ◽  
Jeremy N. Harvey ◽  
Adrian J. Mulholland
Keyword(s):  
Protein Dynamics ◽  
Enzyme Catalysis ◽  
Isotope Effects ◽  
State Theory ◽  
Quantum Tunnelling ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Kinetic Isotope ◽  
Temperature Dependences ◽  
Multiple Conformations

One of the most controversial questions in enzymology today is whether protein dynamics are significant in enzyme catalysis. A particular issue in these debates is the unusual temperature-dependence of some kinetic isotope effects for enzyme-catalysed reactions. In the present paper, we review our recent model [Glowacki, Harvey and Mulholland (2012) Nat. Chem. 4, 169–176] that is capable of reproducing intriguing temperature-dependences of enzyme reactions involving significant quantum tunnelling. This model relies on treating multiple conformations of the enzyme–substrate complex. The results show that direct ‘driving’ motions of proteins are not necessary to explain experimental observations, and show that enzyme reactivity can be understood and accounted for in the framework of transition state theory.

Theoretical Simulations of Heavy-Atom Kinetic Isotope Effects in Aliphatic Claisen Rearrangement

2020 ◽  
Vol 124 (51) ◽  
pp. 10678-10686
Author(s):  
Yuqing Xu ◽  
Kin-Yiu Wong ◽  
Meishan Wang ◽  
Desheng Liu ◽  
Wenkai Zhao ◽  
...  
Keyword(s):  
Claisen Rearrangement ◽  
Isotope Effects ◽  
Heavy Atom ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope ◽  
Theoretical Simulations

Direct dynamics calculations of reaction rate and kinetic isotope effects in enzyme catalysed reactions

2002 ◽  
Vol 122 ◽  
pp. 223-242 ◽  
Author(s):  
Gary Tresadern ◽  
Sara Nunez ◽  
Paul F. Faulder ◽  
Hong Wang ◽  
Ian H. Hillier ◽  
...  
Keyword(s):  
Reaction Rate ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Direct Dynamics ◽  
Kinetic Isotope
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