Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The muscle and heart lactate dehydrogenase (LDHs) of rabbit and pig are specifically cleaved at a single position by HIV-1 protease, resulting in the conversion of 36-kDa subunits of the oligomeric enzymes into 21- and 15-kDa protein bands as analyzed by SDS-PAGE. While the proteolysis was observed at neutral pH, it became more pronounced at pH 6.0 and 5.0. The time courses of the cleavage of the 36-kDa subunits were commensurate with the time-dependent loss of both quaternary structure and enzymatic activity. These results demonstrated that deoligomerization of rabbit muscle LDH at acidic pH rendered its subunits more susceptible to proteolysis, suggesting that a partially denatured form of the enzyme was the actual substrate. Proteolytic cleavage of the rabbit muscle enzyme occurred at a decapeptide sequence, His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp (scissile bond denoted throughout by an asterisk), which constitutes a "strand-loop" element in the muscle and heart LDH structures and contains the active site histidyl residue His-193. The kinetic parameters Km, Vmax/KmEt, and Vmax/Et for rabbit muscle LDH and the synthetic decapeptide Ac-His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp-NH2 were nearly identical, suggesting that the decapeptide within the protein substrate is conformationally mobile, as would be expected for the peptide substrate in solution. Insertion of part of this decapeptide sequence into bacterial galactokinase likewise rendered this protein susceptible to proteolysis by HIV-1 protease, and site-directed mutagenesis of this peptide in galactokinase revealed that the Glu residue at the P2' was important to binding to HIV-1 protease. Crystallographic analysis of HIV-1 protease complexed with a tight-binding peptide analogue inhibitor derived from this decapeptide sequence revealed that the "strand-loop" structure of the protein substrate must adopt a beta-sheet structure upon binding to the protease. The Glu residue in the P2' position of the inhibitor likely forms hydrogen-bonding interactions with both the alpha-amide and gamma-carboxylic groups of Asp-30 in the substrate binding site.
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PMID:Proteolysis of an active site peptide of lactate dehydrogenase by human immunodeficiency virus type 1 protease. 142 Jan 38

The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.
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PMID:Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation. 158 12

A rapid sensitive method for the quantitation in vitro of HIV-1 protease activity has been developed. A fluorogenic compound, N alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta-naphthylamide, which contains Phe-Pro, a dipeptide bond recognized by HIV-1 protease, was used as substrate. The substrate was hydrolyzed by HIV-1 protease into a fluorescent naphthylated product (Pro-MeO-beta-naphthylamide). Fluorescence due to the release of Pro-MeO-beta-naphthylamide was measured continuously by spectrofluorometry. This oligopeptide was found to be a good substrate for HIV-1 protease. The Km and kappa cat for the hydrolysis of N alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta- naphthylamide by HIV-1 protease were calculated to be 2.0 +/- 0.2 mM and 75 +/- 6 s-1, respectively. These values are comparable with those of other natural substrates of HIV-1 protease. The method is highly sensitive, reproducible, and suited to a variety of applications, including the analysis of large numbers of samples for detailed enzymological studies.
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PMID:Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease. 159 89

Analogues of peptides ranging in size from three to six amino acids and containing the hydroxyethylene dipeptide isosteres Phe psi Gly, Phe psi Ala, Phe psi NorVal, Phe psi Leu, and Phe psi Phe, where psi denotes replacement of CONH by (S)-CH(OH)CH2, were synthesized and studied as HIV-1 protease inhibitors. Inhibition constants (Ki) with purified HIV-1 protease depend strongly on the isostere in the order Phe psi Gly greater than Phe psi Ala greater than Phe psi NorVal greater than Phe psi Leu greater than Phe psi Phe and decrease with increasing length of the peptide analogue, converging to a value of 0.4 nM. Ki values are progressively less dependent on inhibitor length as the size of the P1' side chain within the isostere increases. The structures of HIV-1 protease complexed with the inhibitors Ala-Ala-X-Val-Val-OMe, where X is Phe psi Gly, Phe psi Ala, Phe psi NorVal, and Phe psi Phe, have been determined by X-ray crystallography (resolution 2.3-3.2 A). The crystals exhibit symmetry consistent with space group P6(1) with strong noncrystallographic 2-fold symmetry, and the inhibitors all exhibit 2-fold disorder. The inhibitors bind in similar conformations, forming conserved hydrogen bonds with the enzyme. The Phe psi Gly inhibitor adopts an altered conformation that places its P3' valine side chain partially in the hydrophobic S1' pocket, thus suggesting an explanation for the greater dependence of the Ki value on inhibitor length in the Phe psi Gly series. From the kinetic and crystallographic data, a minimal inhibitor model for tight-binding inhibition is derived in which the enzyme subsites S2-S2' are optimally occupied. The Ki values for several compounds are compared with their potencies as inhibitors of proteolytic processing in T-cell cultures chronically infected with HIV-1 (MIC values) and as inhibitors of acute infectivity (IC50 values). There is a rank-order correspondence, but a 20-1000-fold difference, between the values of Ki and those of MIC or IC50. IC50 values can approach those of Ki but are highly dependent on the conditions of the acute infectivity assay and are influenced by physiochemical properties of the inhibitors such as solubility.
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PMID:Hydroxyethylene isostere inhibitors of human immunodeficiency virus-1 protease: structure-activity analysis using enzyme kinetics, X-ray crystallography, and infected T-cell assays. 163 5

The peptidolytic reaction of HIV-1 protease has been investigated by using four oligopeptide substrates, Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, Ac-Ser-Gln-Ser-Tyr-Pro-Val-Val-NH2, and Ac-Arg-Lys-Ile-Leu-Phe-Leu-Asp-Gly-NH2, that resemble two cleavage sites found within the naturally occurring polyprotein substrates Pr55gag and Pr160gag-pol. The values for the kinetic parameters V/KEt and V/Et were 0.16-7.5 mM-1 s-1 and 0.24-29 s-1, respectively, at pH 6.0, 0.2 M NaCl, and 37 degrees C. By use of a variety of inorganic salts, it was concluded that the peptidolytic reaction is nonspecifically activated by increasing ionic strength. V/K increased in an apparently parabolic fashion with increasing ionic strength, while V was either increased or decreased slightly. From product inhibition studies, the kinetic mechanism of the protease is either random or ordered uni-bi, depending on the substrate studied. The reverse reaction or a partial reverse reaction (as measured by isotope exchange of the carboxylic product into substrate) was negligible for most of the oligopeptide substrates, but the enzyme catalyzed the formation of Ac-Ser-Gln-Asn-Tyr-Phe-Leu-Asp-Gly-NH2 from the products Ac-Ser-Gln-Asn-Tyr and Phe-Leu-Asp-Gly-NH2. The protease-catalyzed exchange of an atom of 18O from H2 18O into the re-formed substrates occurred at a rate which was 0.01-0.12 times that of the forward peptidolytic reaction. The results of these studies are in accord with the formation of a kinetically competent enzyme-bound amide hydrate intermediate, the collapse of which is the rate-limiting chemical step in the reaction pathway.
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PMID:Human immunodeficiency virus-1 protease. 1. Initial velocity studies and kinetic characterization of reaction intermediates by 18O isotope exchange. 188 30

We show here for the first time that actin, troponin C, Alzheimer amyloid precursor protein (AAP), and pro-interleukin 1 beta (pro-IL-1 beta), are substrates of the protease encoded by the human immunodeficiency virus (HIV) type-1. As has been seen in other non-viral protein substrates of the HIV protease, the presence of Glu residues in the P2' position appears to play an important role in substrate recognition. Three of the four bonds cleaved in actin, two of the three in troponin C, and all of the bonds hydrolyzed in AAP and pro-IL-1 beta have a P2' Glu residue. In fact, Glu residues are accommodated in all positions from P4 to P4' surrounding the scissile bond in substrates of the HIV proteases, and as many as 4 adjacent Glu residues were seen in one of the bonds cleaved in AAP. This study of non-viral protein substrates has also revealed unexpected amino acids such as Gly, Arg, and Glu in the scissile bond itself rather than the more conventional hydrophobic amino acids. The HIV-2 protease hydrolyzed actin in a manner similar to that of the HIV-1 enzyme, but its cleavage of troponin C was distinct in that it split a bond adjacent to a triplet of Glu residues in P2, P3, and P4 that was refractory to the HIV-1 enzyme. Documentation of cleavage sites in the several important cellular proteins noted above has extended our understanding of the features in a substrate that are recognized by these multi sub-site proteases of retroviral maturation. Moreover, the present work adds to an accumulating body of evidence which demonstrates that these enzymes can damage crucial structural and regulatory cellular proteins if ever their activity is expressed outside the viral particle itself.
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PMID:Actin, troponin C, Alzheimer amyloid precursor protein and pro-interleukin 1 beta as substrates of the protease from human immunodeficiency virus. 190 79

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.
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PMID:Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase. 204 56

Heptapeptide substrates containing a single amino acid substitution at the p2' position of the gag p17-p24 junction (S-Q-N-Y-P-X-V where X = G, A, L, I, F, and W) were compared as substrates for HIV-1 protease. Binding of the Ile-, Leu-, and Ala- containing peptides was about equal although hydrolysis was 20-fold lower for the Ala and Leu peptides compared to Ile. Insertion of Gly or Phe at the p2' position resulted in significantly lower cleavage of the peptide while a Trp-containing peptide was not cleaved. These data suggest that a relatively small hydrophobic amino acid is important for hydrolysis and binding at this site. Structure-activity studies such as those described here may be useful in the design of specific inhibitors for HIV-1 protease.
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PMID:Substitutions at the P2' site of gag p17-p24 affect cleavage efficiency by HIV-1 protease. 218 16

Purified HIV-1 protease hydrolyzes H-Ser-Gln-Asn-Leu-Phe(NO2)-Leu-Asp-Gly-NH2 (Peptide 1) and acetyl-Arg-Lys-Ile-Leu-Phe(NO2)-Leu-Asp-Gly-NH2 (Peptide 2) between the (p-nitro)phenylalanyl and leucyl residues. The cleavage of Peptides 1 and 2 resulted in a decrease in uv absorbance at 310 nm. The HIV-1 protease-catalyzed peptidolysis of Peptides 1 and 2 was characterized by a linear time course at substrate turnover of less than 20%. The solubilities of these substrates at pH 5.0 were sufficient to provide initial rate measurements over a concentration range of 0.05-0.5 mM. Steady-state kinetic data and inhibition constants using both spectrophotometric and high performance liquid chromatography (HPLC) analysis of the peptidolysis of these substrates resulted in comparable values.
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PMID:Chromophoric peptide substrates for the spectrophotometric assay of HIV-1 protease. 218 99

We have used an 'activated' molecular dynamics approach to simulate flap opening in HIV-1 protease. An initial impulse for flap opening was provided by applying harmonic constraints to non-flap residues. After an initial 'melting' phase, the two beta-hairpin structures that constitute the flaps opened to a 25 A gap within 200 ps of simulation. Analysis of backbone torsion angles suggests that flap opening is related to conformational changes at Lys 45, Met 46, Gly 52 and Phe 53. In contrast, similar molecular dynamics simulations on the M46I mutant, which is associated with drug resistance, indicates that this mutation stabilizes the flaps in a closed conformation.
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PMID:Flap opening in HIV-1 protease simulated by 'activated' molecular dynamics. 779 68


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