Is the Critical Role of Loop 3 ofEscherichia coli6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase in Catalysis Due to Loop-3 Residues Arginine-84 and Tryptophan-89? Site-Directed Mutagenesis, Biochemical, and Crystallographic Studies†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (24) ◽  
pp. 8590-8599 ◽  
Author(s):  
Yue Li ◽  
Jaroslaw Blaszczyk ◽  
Yan Wu ◽  
Genbin Shi ◽  
Xinhua Ji ◽  
...  
Keyword(s):  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Critical role of Arg433 in rat transketolase activity as probed by site-directed mutagenesis

1998 ◽  
Vol 333 (2) ◽  
pp. 367-372 ◽  
Author(s):  
Yunjo SOH ◽  
Byoung J. SONG ◽  
Jiingjau JENG ◽  
Abraham T. KALLARAKAL
Keyword(s):  
Critical Role ◽  
Immunoblot Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Hydrogen Bond Interactions ◽  
Apparent Homogeneity ◽  
Arginine Residues ◽  
Biochemical Analyses

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405–2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102 → Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350 → Ala, Arg506 → Ala and Arg433 → Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350 → Ala and Arg433 → Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433 → Ala mutant protein was less stable than the wild-type and Arg350 → Ala proteins at 55 °C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433 → Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433 → Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.

Access of the substrate to the active site of squalene and oxidosqualene cyclases: comparative inhibition, site-directed mutagenesis and homology-modelling studies

2005 ◽  
Vol 33 (5) ◽  
pp. 1202-1205 ◽  
Author(s):  
S. Oliaro-Bosso ◽  
T. Schulz-Gasch ◽  
S. Taramino ◽  
M. Scaldaferri ◽  
F. Viola ◽  
...  
Keyword(s):  
Active Site ◽  
Homology Modelling ◽  
Critical Role ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Specific Substrate ◽  
Oxidosqualene Cyclase ◽  
Active Site Cavity

Substrate access to the active-site cavity of squalene-hopene cyclase from Alicyclobacillus acidocaldarious and lanosterol synthase [OSC (oxidosqualene cyclase)] from Saccharomyces cerevisiae was studied by an inhibition, mutagenesis and homology-modelling approach. Crystal structure and homology modelling indicate that both enzymes possess a narrow constriction that separates an entrance lipophilic channel from the active-site cavity. The role of the constriction as a mobile gate that permits substrate passage was investigated by experiments in which critically located Cys residues, either present in native protein or inserted by site-directed mutagenesis, were labelled with specifically designed thiol-reacting molecules. Some amino acid residues of the yeast enzyme, selected on the basis of sequence alignment and a homology model, were individually replaced by residues bearing side chains of different lengths, charges or hydrophobicities. In some of these mutants, substitution severely reduced enzymatic activity and thermal stability. Homology modelling revealed that in these mutants some critical stabilizing interactions could no longer occur. The possible critical role of entrance channel and constriction in specific substrate recognition by eukaryotic OSC is discussed.

Role of Cysteine Residues in the N-Terminal Extracellular Domain of the Human VIP 1 Receptor for Ligand Binding A Site-directed Mutagenesis Studya

2006 ◽  
Vol 805 (1) ◽  
pp. 585-589 ◽  
Author(s):  
PASCALE GAUDIN ◽  
ALAIN COUVINEAU ◽  
JEAN-JOSÉ MAORET ◽  
CHRISTIANE ROUYER-FESSARD ◽  
MARC LABURTHE
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
A Site

scholarly journals Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.

1992 ◽  
Vol 267 (36) ◽  
pp. 25754-25758
Author(s):  
J Wu ◽  
D Filer ◽  
A.J. Friedhoff ◽  
M Goldstein
Keyword(s):  
Tyrosine Hydroxylase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals The role of cysteines in polyketide synthases. Site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways

1991 ◽  
Vol 266 (15) ◽  
pp. 9971-9976 ◽  
Author(s):  
T. Lanz ◽  
S. Tropf ◽  
F.J. Marner ◽  
J. Schröder ◽  
G. Schröder
Keyword(s):  
Polyketide Synthases ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Key Enzymes

scholarly journals The role of Val68(E11) in ligand binding to sperm whale myoglobin. Site-directed mutagenesis of a synthetic gene.

1990 ◽  
Vol 265 (20) ◽  
pp. 11788-11795
Author(s):  
K D Egeberg ◽  
B A Springer ◽  
S G Sligar ◽  
T E Carver ◽  
R J Rohlfs ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Sperm Whale ◽  
Site Directed Mutagenesis ◽  
Synthetic Gene ◽  
Directed Mutagenesis ◽  
Sperm Whale Myoglobin

Probing the Role of Tyr 64 ofTreponema denticolaCystalysin by Site-Directed Mutagenesis and Kinetic Studies†

Biochemistry ◽  
2005 ◽  
Vol 44 (42) ◽  
pp. 13970-13980 ◽  
Author(s):  
Barbara Cellini ◽  
Mariarita Bertoldi ◽  
Riccardo Montioli ◽  
Carla Borri Voltattorni
Keyword(s):  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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