Studies on the role of the S₄ substrate binding site of HIV proteinases

Abstract

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,

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

“…Indeed, the specificity constants for the ACC substrates are at least 3 orders of magnitude lower than those for longer substrates. That ACC proteolysis is measurable runs contrary to the findings of prior studies that have shown the minimal length of an HIV PR substrate to be P4 to P3Ј (10,42). However, specificity constants this low would be extremely difficult to measure using the detection methods typically used with HIV PR, which often involve high-pressure liquid chromatographic analysis and/or the use of a less-sensitive chromogenic or fluorogenic reporter group.…”

“…However, all three libraries showed a strong tendency toward small residues. Similarly, a preference for small amino acids such as serine in this position has been reported (42).…”

Studies on the Symmetry and Sequence Context Dependence of the HIV-1 Proteinase Specificity

“…It is important to note that this shorter peptide showed 10-fold higher k cat and k cat /K m values than peptide 16. The effect of residues P 5 and P 4 on substrate hydrolysis has been reported previously (11,23). To compare the specificity of the S 3 and S 3 Ј substrate binding pockets in an identical sequence context, the Asp residues of the palindromic peptide 17 were substituted to Gly, Gln, Phe and Leu residues (Table III).…”

“…In addition, peptide 2 also contains Thr at both P 2 and P 2 Ј positions. Furthermore, peptides 1 and 2 with the exception of the P 2 Ј residues share the same sequence in the P 4 -P 3 Ј region, which is the major determinant for specificity (23). However, peptide 2 was found to be a much poorer substrate of the HIV proteinases than peptide 1 (see Table I and Ref.…”

“…We have also compared the specificity of the HIV-1 and HIV-2 proteinases using a series of oligopeptide substrates containing single amino acid substitutions in the sequence of SP-211 (see peptide 3 in Table I), a peptide that corresponds to the type 1 MA/CA cleavage site in HIV-1 (10,23). In these studies it was found that substitution of Pro at the P 1 Ј position to any other amino acid tested, including Tyr, formed nonhydrolyzable or very poor substrates of HIV proteinases.…”