title: Programming the Rous sarcoma virus protease to cleave new substrate sequences.
abstract: The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: T W
authors.initials: TW
authors.lastname: Ridky
authors.forename: D
authors.initials: D
authors.lastname: Bizub-Bender
authors.forename: C E
authors.initials: CE
authors.lastname: Cameron
authors.forename: I T
authors.initials: IT
authors.lastname: Weber
authors.forename: A
authors.initials: A
authors.lastname: Wlodawer
authors.forename: T
authors.initials: T
authors.lastname: Copeland
authors.forename: A M
authors.initials: AM
authors.lastname: Skalka
authors.forename: J
authors.initials: J
authors.lastname: Leis
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000201:enzymology; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D016282:Aspartic Acid Endopeptidases; C072891:Rous sarcoma virus protease; C077822:kynostatin 272