scholarly journals An analysis of reaction pathways for proton tunnelling in methylamine dehydrogenase

2006 ◽  
Vol 361 (1472) ◽  
pp. 1387-1398 ◽  
Author(s):  
Sara Nuñez ◽  
Gary Tresadern ◽  
Ian H Hillier ◽  
Neil A Burton
Keyword(s):  
Triosephosphate Isomerase ◽  
Bond Cleavage ◽  
Isotope Effects ◽  
Enzymatic Reactions ◽  
Amino Acid Residues ◽  
Methylamine Dehydrogenase ◽  
Specific Amino Acid ◽  
Quantum Tunnelling ◽  
Kinetic Isotope ◽  
Semi Empirical

Computational methods have now become a valuable tool to understand the way in which enzymes catalyse chemical reactions and to aid the interpretation of a diverse set of experimental data. This study focuses on the influence of the condensed-phase environment structure on proton transfer mechanisms, with an aim to understand how C–H bond cleavage is mediated in enzymatic reactions. We shall use a combination of molecular simulation, ab initio or semi-empirical quantum chemistry and semi-classical multidimensional tunnelling methods to consider the primary kinetic isotope effects of the enzyme methylamine dehydrogenase (MADH), with reference to an analogous application to triosephosphate isomerase. Analysis of potentially reactive conformations of the system, and correlation with experimental isotope effects, have highlighted that a quantum tunnelling mechanism in MADH may be modulated by specific amino acid residues, such as Asp428, Thr474 and Asp384.

Transition-state structure for the hydronium-ion-promoted hydrolysis of α-d-glucopyranosyl fluoride

2015 ◽  
Vol 93 (4) ◽  
pp. 463-467 ◽  
Author(s):  
Jefferson Chan ◽  
Ariel Tang ◽  
Andrew J. Bennet
Keyword(s):  
Transition State ◽  
Kinetic Isotope Effect ◽  
Bond Cleavage ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Theoretical Modelling ◽  
Kinetic Isotope ◽  
Hydronium Ion ◽  
Anomeric Carbon ◽  
Hydrolysis Of

The transition state for the hydronium-ion-promoted hydrolysis of α-d-glucopyranosyl fluoride in water has been characterized by combining multiple kinetic isotope effect measurements with theoretical modelling. The measured kinetic isotope effects for the C1-deuterium, C2-deuterium, C5-deuterium, anomeric carbon-13, and ring oxygen-18 are 1.219 ± 0.021, 1.099 ± 0.024, 0.976 ± 0.014, 1.014 ± 0.005, and 0.991 ± 0.013, respectively. The transition state for the hydronium ion reaction is late with respect to both C–F bond cleavage and proton transfer.

Deuterium kinetic isotope effects on the thermal isomerizations of deuteriocyclopropane to deuterium-labeled propenes

2005 ◽  
Vol 83 (9) ◽  
pp. 1510-1515
Author(s):  
John E Baldwin ◽  
Stephanie R Singer
Keyword(s):  
Key Words ◽  
Gas Phase ◽  
Bond Cleavage ◽  
Isotope Effects ◽  
Reactive Intermediates ◽  
Kinetic Isotope Effects ◽  
Thermal Rearrangement ◽  
Carbon Bond ◽  
Kinetic Isotope ◽  
Carbon Carbon Bond

The gas-phase thermal isomerizations of deuteriocyclopropane to the four possible monodeuterium-labeled propenes have been followed at 435 °C. The observed distribution of products provides estimates of two deuterium kinetic isotope effects, the secondary [Formula: see text] for the carbon–carbon bond cleavage leading to trimethylene diradical reactive intermediates and the primary [Formula: see text] ratio for a [1,2] shift of a hydrogen or deuterium leading from the diradical to a labeled propene. The values determined are [Formula: see text] = 1.09 ± 0.03 and [Formula: see text] = 1.55 ± 0.06. The experimental [Formula: see text] value found agrees well with some, but not all, earlier calculated values and conjectures. Key words: cyclopropane, thermal rearrangement, kinetic isotope effects.

Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal ω-3 fatty acid desaturases

2000 ◽  
Vol 28 (6) ◽  
pp. 632-635 ◽  
Author(s):  
D. Meesapyodsuk ◽  
D. W. Reed ◽  
C. K. Savile ◽  
P. H. Buist ◽  
U. A. Schäfer ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Bond Cleavage ◽  
Isotope Effects ◽  
Fatty Acid Desaturases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Kinetic Isotope ◽  
Acid Analogue ◽  
And Function

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the ω-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16–20 carbons with a preference for ω-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of ω-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C18 fatty acid analogue were measured via competitive incubation experiments. Whereas kH/kD at the ω-3 position was shown to be large, essentially no kinetic isotope effect at the ω-2 position was observed for the plant or the nematode enzymes. These results indicate that ω-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.

Carbon-bromine bond cleavage – A perspective from bromine and carbon kinetic isotope effects on model debromination reactions

Chemosphere ◽  
2018 ◽  
Vol 193 ◽  
pp. 17-23 ◽  
Author(s):  
Rabindra Nath Manna ◽  
Anna Grzybkowska ◽  
Faina Gelman ◽  
Agnieszka Dybala-Defratyka
Keyword(s):  
Bond Cleavage ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope

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.

scholarly journals Catalytic mechanism of a family 3 β-glucosidase and mutagenesis study on residue Asp-247

2001 ◽  
Vol 355 (3) ◽  
pp. 835-840 ◽  
Author(s):  
Yaw-Kuen LI ◽  
Jiunly CHIR ◽  
Fong-Yi CHEN
Keyword(s):  
Amino Acid ◽  
Essential Amino Acid ◽  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Kinetic Isotope

A family 3 β-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the kcat values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl–enzyme intermediate. The large Bronsted constant (β =-0.85) for the leaving-group-dependent portion (pKa of leaving phenols > 7) indicates substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl β-D-glucopyanoside, o-nitrophenyl β-D-glucopyanoside and p-cyanophenyl β-D-glucopyanoside as substrates were 1.17±0.02, 1.19±0.02 and 1.04±0.02 respectively. These results support an SN1-like mechanism for the deglucosylation step and an SN2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (kcat/Km) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20% of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.

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