Unmasking of Deuterium Kinetic Isotope Effects on the Methane Monooxygenase Compound Q Reaction by Site-Directed Mutagenesis of Component B

2001 ◽  
Vol 123 (42) ◽  
pp. 10421-10422 ◽  
Author(s):  
Brian J. Brazeau ◽  
Bradley J. Wallar ◽  
John D. Lipscomb
Keyword(s):  
Isotope Effects ◽  
Methane Monooxygenase ◽  
Kinetic Isotope Effects ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Isotope ◽  
Compound Q

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.

scholarly journals Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase

2000 ◽  
Vol 276 (7) ◽  
pp. 4549-4553 ◽  
Author(s):  
Shannon S. Stahl ◽  
Wilson A. Francisco ◽  
Maarten Merkx ◽  
Judith P. Klinman ◽  
Stephen J. Lippard
Keyword(s):  
Isotope Effects ◽  
Methane Monooxygenase ◽  
Kinetic Isotope Effects ◽  
Soluble Methane Monooxygenase ◽  
Kinetic Isotope

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
Sign in / Sign up

Export Citation Format

Share Document