title: Interactions of substrates and inhibitors with a family of tethered HIV-1 and HIV-2 homo- and heterodimeric proteinases.
abstract: Genes were constructed to encode single-chain tethered human immunodeficiency virus HIV-1/HIV-1 and HIV-2/HIV-2 homodimeric proteinases and two HIV-1/HIV-2 heterodimers which differed in the nature of the interface strands. All four constructs under the control of a heat-inducible promoter were expressed in E. coli and the resultant proteinases were purified therefrom. Kinetic parameters (Km, kcat and kcat/Km) were derived for the interaction of the tethered homo and heterodimeric proteinases with two distinct substrates at a variety of pH values. All four enzymes were comparably active toward one substrate. With the second substrate at pH 4.7, the kcat/Km value was best for the HIV-1/1 tethered homodimer, 15-fold lower for the two heterodimeric proteinases, and was reduced by an additional 6-fold for the HIV-2/2 homodimer. From the Ki values determined for the interactions of the four tethered dimer proteinases with a systematic series of synthetic inhibitors, a parallel trend was observed. Whereas several inhibitors were equipotent against all four enzymes, two were discriminatory in that they inhibited strongly the HIV-1/1 homodimer and the two heterodimeric proteinases but had little effect on the HIV -2/2 tethered homodimer (or its untethered wild-type counterpart from HIV-2). The significance of these findings for active site interaction with HIV-proteinases is considered.
authors.affiliation: Department of Biochemistry, University of Wales College of Cardiff, United Kingdom.
authors.forename: J T
authors.initials: JT
authors.lastname: Griffiths
authors.forename: L A
authors.initials: LA
authors.lastname: Tomchak
authors.forename: J S
authors.initials: JS
authors.lastname: Mills
authors.forename: M C
authors.initials: MC
authors.lastname: Graves
authors.forename: N D
authors.initials: ND
authors.lastname: Cook
authors.forename: B M
authors.initials: BM
authors.lastname: Dunn
authors.forename: J
authors.initials: J
authors.lastname: Kay
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D016333:HIV Protease / Q000096:biosynthesis / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D015497:HIV-1 / Q000201:enzymology*; D015498:HIV-2 / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D017320:HIV Protease Inhibitors; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease; C065184:p16 protease, Human immunodeficiency virus 2