DIFFERENT REQUIREMENTS FOR PRODUCTIVE INTERACTION BETWEEN THE ACTIVE-SITE OF HIV-1 PROTEINASE AND SUBSTRATES CONTAINING -HYDROPHOBIC-ASTERISK HYDROPHOBIC- OR -AROMATIC-ASTERISK PRO- CLEAVAGE SITES

The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kezdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic-type produces a peptide cleaved very efficiently (k(cat) > 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenyl-alanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis. Thus, ability of HIV-1 PR to selectively and efficiently cleave a variety of totally different sequences may be derived, in part, from extensive interactions at long distances from the actual scissile peptide bond and the inherent flexibility of several key loops of polypeptide structure of the enzyme.