INTERACTIONS OF SUBSTRATES AND INHIBITORS WITH A FAMILY HIV-1 AND HIV-2 HOMODIMERIC AND HETERODIMERIC PROTEINASES

Genes were constructed to encode single-chain tethered human immunodef iciency 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 inte rface strands, Ah four constructs under the control of a heat-inducibl e promoter were expressed in E. coli and the resultant proteinases wer e purified therefrom. Kinetic parameters (K-m, k(cat) and k(cat)/K-m) were derived for the interaction of the tethered homo and heterodimeri c 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 k(cat)/K-m value was best for the HIV -1/1 tethered homodimer, 15-fold lower for the two heterodimeric prote inases, and was reduced by an additional 6-fold for the HIV-2/2 homodi mer. From the K-i values determined for the interactions of the four t ethered dimer proteinases with a systematic series of synthetic inhibi tors, a parallel trend was observed. Whereas several inhibitors were e quipotent against all four enzymes, two were discriminatory in that th ey inhibited strongly the HIV-1/1 homodimer and the two heterodimeric proteinases but had little effect on the HIV-2/2 tethered homodimer (o r its untethered wild-type counterpart from HIV-2). The significance o f these findings for active site interaction with HIV-proteinases is c onsidered.