The xutrx~r~ spa5tkity af rho humn inmunodclicicncy vitua gyp I (HIV-I) unci ~ypc' 2 (HIV-2) pruteimscr ww cumpsrrcl uxiny oli~sprptidcx carreqxMin@ to ckrtveyc aitcx in tha Gqt und Gug4W palyprutcias ur both viruses. All p6lxidcI mimickinp clcavtiyo ailcr MI the junction QT major functiunul pratcin danr;rins were correctly elctivcd by both cnxymcs. Hawuvrr, some athcr peptidcs thought tm reprrwcnt rrcandary cle~ugl: sites rcmiiierd intact. 'Ptrc kin& p:trcttnctcrx (A'* end k,,) abrainrd far the difikranc atbstrstu rhuwal wcvcrd hunJrc&fultl variation but were rimibr for the MTIC subrtrutc.
Comparison of the three-dimensional structure of bovine chymosin with the structures of homologous aspartic proteinases complexed with peptide inhibitors shows that Val111 in chymosin occupies a position between the specificity subsites S1 and S3. A mutation corresponding to Val111 to Phe has been introduced in an intermediary plasmid construct of prochymosin by bridging its unique restriction sites by a synthetic mutant oligonucleotide duplex. A prochymosin fusion product was expressed in Escherichia coli in such a way that the extension and substitution of the propart does not interfere with the activation of the zymogen. After activation of the crude prochymosin, the enzyme was purified by affinity chromatography on Sepharose with V-dL-P-F-F-V-dL as ligand. This procedure provided large amounts of pure protein as judged by FPLC, the activity/protein ratio, and SDS-PAGE. The enzymatic properties were determined by using a variety of peptide substrates and inhibitors; KM values for the mutant enzyme were approximately twice those of the wild type, but the kcat values were little changed. The mutant enzyme was crystallized, X-ray data were collected to 2.0-A resolution by using a FAST area detector, and the structure was solved by using difference Fourier methods and refined to an R factor of 19.5%. The mutation leads to only local changes in conformation, with the phenylalanine side chain occupying part of the S1 and S3 pockets. This accounts for the increased KM of this mutant for a substrate with a large phenylalanine side chain at P1. It is also consistent with the higher affinity of the mutant for an inhibitor with small side chains at P1 and P3 when compared with the wild-type enzyme.
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.
Kinetic una )'sis of the hydrtd~sis of the I~ptide lI.Val,Ser.GIn,Asn.Tyr*Pro.ll¢.VaI.GIn.Nll~ and its analo$,t obtainctl by varyinll the !ength and introduein= substitutions at the P, site was carried out with t~flh HIV.I and ItlV.2 prolein~se~. Deletion or ih¢ t~rmlnal Vtd ~nd Gin had only metier|tie effect on ilia ~uhstrltte hydroly~h, while the d~lation of tli.c P.~. Set ~ts well as P; V,I Ilrcatly reduwd the substrata hydrolysis. Thi~ is pr~dicled to bc due to the loss of Intera,:tions betwoen main ~h~ins of the en,~yme ~ntl thtt substrata. $=bstitution of tile P,~ Ser h:,, amino =raids having high frequency 0f occurrenc~ in//turns resulted in llood substratcs, while larlle ~mino lteids were unfavorable in thi~ position. The two prntai=mses acted similarly, except for substrates havini! TItr, Val and Let= substitutions, which were I~=iler ,o;ommodated in the FIlV.2 substrata bindin$ pocket,
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