Modification of the pH profile of chymosin using site-directed mutagenesis

1989 ◽  
Vol 17 (4) ◽  
pp. 763-764 ◽  
Author(s):  
DIMITRIS MANTAFOUNIS ◽  
JAMES E. PITTS
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ph Profile

scholarly journals Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis

1995 ◽  
Vol 309 (1) ◽  
pp. 341-346 ◽  
Author(s):  
M H Rider ◽  
K M Crepin ◽  
M De Cloedt ◽  
L Bertrand ◽  
D Vertommen ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Guanidinium Chloride ◽  
Wild Type Enzyme ◽  
Ph Profile ◽  
Substrate Concentrations

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis

Biochemistry ◽  
1992 ◽  
Vol 31 (24) ◽  
pp. 5467-5471 ◽  
Author(s):  
Herman Van Tilbeurgh ◽  
John Jenkins ◽  
Mohammed Chiadmi ◽  
Joel Janin ◽  
Shoshana J. Wodak ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Glucose Isomerase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ph Profile ◽  
Metal Specificity

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
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