Enzymology, kinetics and synthetic, physical and analytical chemistry are being used to investigate the mechanisms of catalysis by and assembly of the reverse transcriptase (RT) and protease of human immunodeficiency virus type 1 (HIV-1), with the ultimate goal of developing specific inhibitors for these enzymes. i) In a previous report, we described an amino-acid repeat motif, resembling a modified """"""""leucine zipper"""""""", in the RT of several HIV-1 and HIV-2 isolates, which we suggested might be involved in subunit association. X-ray studies by Kohlstaedt et al. have now indicated that the regions containing this motif in the p51 and p66 subunits are not in contact in the crystalline heterodimer. Thus, the function of this highly conserved sequence is as yet undefined. ii) Studies on the acceleration by sodium chloride of the rate of peptide hy- drolysis catalyzed by retroviral proteases as well as by the model mammalian enzyme, pepsin, are now complete. This salt effect, which is almost exclusively on the Michaelis constant, is suggested to result from the enhancement of hydrophobic interactions between the substrate and the enzyme's active site. Using a spectrophotometric assay, which facilitated assessment of substrate solubility, we have observed a monotonic increase in rate with NaCl concentrations up to 5 M. Literature reports of a bell- shaped dependence of the rate on salt concentration presumably result from artifacts due to the failure to detect reduced solubility of the substrate at high salt concentrations. iii) Kinetic studies on the autoprocessing of constructs of the HIV-1 protease containing flanking Pol region sequences and expressed as fusion proteins with the maltose-binding protein of Escherichia coli are in progress. Preliminary results suggest an initial loss of the N-terminal sequence containing the maltose-binding protein to give a 13.2-kDa intermediate which retains the C-terminal Pol sequence; this intermediate is cleaved to the 11-kDa protease in a slower step. At the concentrations used, the initial cleavage is first-order in protein concentration, consistent with a rapid and favorable dimerization of the fusion protein followed by intramolecular proteolysis.
