PMID: 2501781May 1, 1989Paper

Alteration of catalytic properties of chymosin by site-directed mutagenesis

Protein Engineering
J SuzukiT Beppu

Abstract

Artificial mutations of chymosin by recombinant DNA techniques were generated to analyze the structure--function relationship in this characteristic aspartic proteinase. In order to prepare the mutant enzymes in their active form, we established procedures for purification of correctly refolded prochymosin from inclusion bodies produced in Escherichia coli transformants and for its subsequent activation. Mutagenesis by linker insertion into cDNA produced several mutants with an altered ratio of milk clotting activity to proteolytic activity and a different extent of stability. In addition to these mutants, several mutants with a single amino acid exchange were also constructed by site-directed mutagenesis and kinetic parameters of these mutant enzymes were determined by using synthetic hexa- and octa-peptides as substrates. Exchange of Tyr75 on the flap of the enzyme to Phe caused a marked change of substrate specificity due to the change of kcat or Km, depending on the substrate used. Exchange of Val110 and Phe111 also caused a change of kinetic parameters, which indicates functional involvement of these hydrophobic residues in both the catalytic function and substrate binding. The mutant Lys220----Leu showed a marked shift of...Continue Reading

Citations

Jan 1, 1994·Biotechnology Advances·B E Fischer
Aug 1, 1994·Current Opinion in Biotechnology·J Tang, X Lin
Jun 1, 1996·The International Journal of Biochemistry & Cell Biology·G HouenB Foltmann
Apr 1, 1994·Zentralblatt für Veterinärmedizin. Reihe A·M Wiśliński, M Popielarz
Jan 31, 2002·American Journal of Respiratory and Critical Care Medicine·Anna PomésVenugopal Dhanaraj
Jun 8, 2010·Critical Reviews in Biotechnology·A KumarV K Batish
Jan 1, 1990·Annals of the New York Academy of Sciences·T Beppu
Nov 6, 2007·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Shaozhen FengHaoran Lin
May 19, 2011·Proteins·Steven A Spronk, Heather A Carlson
Nov 1, 1993·Molecular & General Genetics : MGG·K MurakamiT Beppu

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