Interactions of substrates and inhibitors with a family of tethered HIV-1 and HIV-2 homo- and heterodimeric proteinases

• Published in: The Journal of biological chemistry 1994-02, Vol.269 (7), p.4787-4793
• Main Authors: GRIFFITHS, J. T, TOMCHAK, L. A, MILLS, J. S, GRAVES, M. C, COOK, N. D, DUNN, B. M, KAY, J
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Aspartic Acid Endopeptidases - biosynthesis; Aspartic Acid Endopeptidases - metabolism; Biological and medical sciences; Cloning, Molecular; Enzymes and enzyme inhibitors; Escherichia coli; Fundamental and applied biological sciences. Psychology; HIV Protease - biosynthesis; HIV Protease - metabolism; HIV Protease Inhibitors - metabolism; HIV Protease Inhibitors - pharmacology; HIV-1 - enzymology; HIV-2 - enzymology; human immunodeficiency virus 1; human immunodeficiency virus 2; Hydrolases; Kinetics; Macromolecular Substances; Molecular Sequence Data; Plasmids; Polymerase Chain Reaction; Protein Multimerization; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37613-5

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.