Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II

2022 ◽  
Author(s):  
Daniel Bím ◽  
Michal Navrátil ◽  
Ondrej Gutten ◽  
Jan Konvalinka ◽  
Zsófia Kutil ◽  
...  
Keyword(s):  
Enzyme Kinetics ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Glutamate Carboxypeptidase Ii ◽  
Glutamate Carboxypeptidase

Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis

FEBS Journal ◽  
2007 ◽  
Vol 274 (18) ◽  
pp. 4731-4741 ◽  
Author(s):  
Petra Mlčochová ◽  
Anna Plechanovová ◽  
Cyril Bařinka ◽  
Daruka Mahadevan ◽  
Jose W. Saldanha ◽  
...  
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Glutamate Carboxypeptidase Ii ◽  
Glutamate Carboxypeptidase

Site-Directed Mutagenesis of Predicted Active Site Residues in Glutamate Carboxypeptidase II

1999 ◽  
Vol 55 (1) ◽  
pp. 179-185 ◽  
Author(s):  
Henry S. Speno ◽  
Ruth Luthi-Carter ◽  
Wendy L. Macias ◽  
Stacey L. Valentine ◽  
Amit R. T. Joshi ◽  
...  
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Glutamate Carboxypeptidase Ii ◽  
Glutamate Carboxypeptidase

scholarly journals Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

2006 ◽  
Vol 395 (3) ◽  
pp. 509-515 ◽  
Author(s):  
Zhong-liang Zheng ◽  
Mao-qing Ye ◽  
Zhen-yu Zuo ◽  
Zhi-gang Liu ◽  
Keng-chang Tai ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Hydrogen Bonds ◽  
Transition State ◽  
Enzyme Kinetics ◽  
Structural Model ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Directed Mutagenesis

Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33→Ala33, Asp60→Ala60, Ser62→Ala62, and Thr220→Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (ΔΔGT). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the Km values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations.

The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

2013 ◽  
Vol 37 (3) ◽  
pp. 330
Author(s):  
Yanan WANG ◽  
Xudong LIU ◽  
Linlin MU ◽  
Zhipeng LIU ◽  
Chunmei LI ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Cynoglossus Semilaevis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Site-directed mutagenesis on alkaline phosphatasefrompseudomonas aeruginosapao1 for enhanced activity

2016 ◽  
Vol 7 (4) ◽  
Author(s):  
UMA SELVARAJ ◽  
THIRUMALAI MUTHUKUMARESAN ◽  
GAYATHRI VIJAYENDRAN ◽  
SENTHIL KUMAR DEVAN ◽  
VENU BABU P ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Enhanced Activity
Sign in / Sign up

Export Citation Format

Share Document