title: Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin.
abstract: The loop exchange mutant chymosin 155-164 rhizopuspepsin was expressed in Trichoderma reesei and exported into the medium to yield a correctly folded and active product. The biochemical characterization and crystal structure determination at 2.5 A resolution confirm that the mutant enzyme adopts a native fold. However, the conformation of the mutated loop is unlike that in native rhizopuspepsin and involves the chelation of a water molecule in the loop. Kinetic analysis using two synthetic peptide substrates (six and 15 residues long) and the natural substrate, milk, revealed a reduction in the activity of the mutant enzyme with respect to the native when acting on both the long peptide substrate and milk. This may be a consequence of the different charge distribution of the mutated loop, its increased size and/or its different conformation.
authors.affiliation: Department of Crystallography, Birkbeck College, University of London, UK.
authors.forename: P G
authors.initials: PG
authors.lastname: Nugent
authors.forename: A
authors.initials: A
authors.lastname: Albert
authors.forename: P
authors.initials: P
authors.lastname: Orprayoon
authors.forename: J
authors.initials: J
authors.lastname: Wilsher
authors.forename: J E
authors.initials: JE
authors.lastname: Pitts
authors.forename: T L
authors.initials: TL
authors.lastname: Blundell
authors.forename: V
authors.initials: V
authors.lastname: Dhanaraj
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D012085:Chymosin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009391:Nephelometry and Turbidimetry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C035794:rhizopuspepsin; D012085:Chymosin