title: Multidisciplinary cycles for protein engineering: site-directed mutagenesis and X-ray structural studies of aspartic proteinases.
abstract: The specificity and pH profile of aspartic proteinases have evolved to include not only pepsin with a broad specificity and an optimal activity in acid media, but also renin, with high specificity for angiotensinogen and activity close to neutral pH. Comparisons of the structures and catalytic activities of aspartic proteinases provide helpful clues for engineering new activity profiles. We illustrate an approach that involves recombinant DNA techniques, biochemistry, structure determination and biocomputing. We use the 3-D structures of inhibitor complexes of several aspartic proteinases to define likely intermediates and specificity sub-sites. The multidisciplinary research is organised as cycles, in which each cycle tests a design hypothesis proposed in the previous cycle. We use one member of the aspartic proteinase family, chymosin, to illustrate these ideas in engineering enzymes with altered pH optima and specificities.
authors.affiliation: Department of Crystallography, Birkbeck College, London, UK.
authors.forename: J E
authors.initials: JE
authors.lastname: Pitts
authors.forename: V
authors.initials: V
authors.lastname: Dhanaraj
authors.forename: C G
authors.initials: CG
authors.lastname: Dealwis
authors.forename: D
authors.initials: D
authors.lastname: Mantafounis
authors.forename: P
authors.initials: P
authors.lastname: Nugent
authors.forename: P
authors.initials: P
authors.lastname: Orprayoon
authors.forename: J B
authors.initials: JB
authors.lastname: Cooper
authors.forename: M
authors.initials: M
authors.lastname: Newman
authors.forename: T L
authors.initials: TL
authors.lastname: Blundell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D014961:X-Ray Diffraction
chemical_list: D016282:Aspartic Acid Endopeptidases