Kinetic and modeling studies of S3-S3' subsites of HIV proteinases

Abstract

Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino acids at P2, P1, P2', and P3' positions (Ala, Leu, Met, Phe). Various amino acids seemed to be acceptable at P3 and P3' positions, while the P2 and P2' positions seemed to be more restrictive. Polar uncharged residues resulted in relatively good binding at P3 and P2 positions, while at P2' and P3' positions they gave very high Km values, indicating substantial differences in the respective S and S' subsites of the enzyme. Lys prevented substrate hydrolysis at any of the P2-P2' positions. The large differences for subsite preference at P2 and P2' positions seem to be at least partially due to the different internal interactions of P2 residue with P1', and P2' residue with P1. As expected on the basis of amino acid frequency in the naturally occurring cleavage sites, hydrophobic residues at P1 position resulted in cleavable peptides, while polar and beta-branched amino acids prevented hydrolysis. On the other hand, changing the P1' Pro to other amino acids prevented substrate hydrolysis, even if the substituted amino acid had produced a good substrate in other oligopeptides representing naturally occurring cleavage sites. The results suggest that the subsite specificity of the HIV proteinases may strongly depend on the sequence context of the substrate.

Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease

“…3), and Partin et al (34) observed that Phe readily substituted for Tyr at this site. Both increased and decreased rates of cleavage for a P1 Phe have been reported for equivalent peptide substrates of the MA/CA site (3,54).…”

“…The amino acids flanking the target scissile bond are generally hydrophobic (16,33,37). A number of studies have used peptide or Gag substrates in an attempt to define the role of specific amino acids at different positions flanking the scissile bond as they impact the rate of cleavage by the viral protease (3,5,13,14,19,30,34,36,38,44,45,51,(53)(54)(55); recently reviewed in reference 27). In the present study we have examined the ability of approximately 15 different amino acids in the P1 position of the five different Gag cleavage sites to support cleavage by the HIV-1 protease (the P1 position is the amino acid immediately upstream of the scissile bond, and the P1Ј position is the amino acid immediately downstream of the scissile bond).…”

A Look inside HIV Resistance through Retroviral Protease Interaction Maps

“…The prediction error for the cleavage rates was less than one log(k cat /K m ) unit for 68% of the protease-substrate pairs; the correlation for the a priori predicted rates versus the experimentally determined rates yielding a correlation coefficient r ¼ 0. 47 (Table S1), but excluded all data for the proteases of one retroviral strain, one at a time, and uses the model to predict the excluded data. Blue bullets correspond to prediction of cleavage activity for new proteases and new substrates (i.e., the cases in which neither the protease nor the peptide was represented in the dataset used in creation of the model).…”

Point mutation in avian sarcoma leukaemia virus protease which increases its activity but impairs infectious virus production

“…The dissolved proteins were fractionated by reverse-phase high-pressure liquid chromatography (RP-HPLC) on a 19x 150mm g-Bondapack C18 column (Waters Associates) with a linear gradient of 0.60% acetonitrile in the presence of 0.05 % TFA in water as previously described (Copeland & Oroszlan, 1988). Fractions from the RP-HPLC column were lyophilized, dissolved in 2 M-guanidine hydrochloride and 100 mM-Tris-HC1 pH 8.0 at a concentration of 200-300 gg/ml, and refolded as previously described (Tozser et al, 1992). Briefly, one volume of protein solution was diluted with two volumes of refolding buffer (20 mM-PIPES pH 7.0, 100 mM-NaC1, 10% glycerol, 1 mM-EDTA).…”