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 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

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

Highly purified, recombinant preparations of the virally encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to 1) their specificities toward non-viral protein and synthetic peptide substrates, and 2) their inhibition by several P1-P1' pseudodipeptidyl-modified substrate analogs. Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys-PE40, is qualitatively the same for HIV-2 protease as published earlier for the HIV-1 enzyme (Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., FitzGerald, D. J., Chaudhary, V. K., Pastan, I., and Heinrikson, R. L. (1990) J. Biol. Chem. 265, 408-413). However, the rates of cleavage at these two sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond. The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides. Hydrolysis by the two proteases of other synthetic peptides modeled after processing sites in HIV-1 and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity. For example, while the two proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-1 gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-1 protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template. Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-1 enzyme; it is better able to hydrolyze substrates with small amino acids in P1 and P1', but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions. In addition to these comparisons of the two proteases with respect to substrate specificity, we present inhibitor structure-activity data for the HIV-2 protease. Relative to P1-P1' statine or Phe psi [CH2N]Pro-modified pseudopeptidyl inhibitors, compounds having Xaa psi[CH(OH)CH2]Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding from 10 to 100 times stronger to HIV-1 protease than to the HIV-2 enzyme. Molecular modeling comparisons based upon the sequence homology of the two enzymes and x-ray crystal structures of HIV-1 protease suggest that most of the nonconservative amino acid replacements occur in regions well outside the catalytic cleft, while only subtle structural differences exist within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2. 220 91

The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E. coli. Single amino acid substitutions (Arg 87 - greater than Lys; Arg 87 - greater than Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone. The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes. The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein.
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PMID:Substitution mutations of the highly conserved arginine 87 of HIV-1 protease result in loss of proteolytic activity. 267 52

Considerable interest exists in the HIV-1 protease for biochemical studies as a potential therapeutic target of acquired immunodeficiency syndrome. We have produced the retroviral enzyme in E. coli from a synthetic gene encoding the protease that was constructed by assembling six overlapping and complementary oligonucleotides into the vector pKK223-3. When expressed in E. coli, the recombinant protease was able to correctly process the HIV-1 core protein p24 from a beta-galactosidase-gag fusion protein and to use a heptapeptide as a substrate for proteolytic cleavage. A single base pair mutation was identified in a recombinant that resulted in the substitution of lysine for asparagine at position 88 and a significant loss of enzyme activity. Through site-directed mutagenesis, the Asn88 was changed to five other residues representative of all classes of amino acids. The correlation between enzyme activity and amino acid substitution suggests that the protease domain surrounding position 88 affects the protein's potential for forming an active homodimeric protein and hence, indicates a biochemical interaction that could be inhibited by novel antiviral compounds.
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PMID:HIV-1 protease: mutagenesis of asparagine 88 indicates a domain required for dimer formation. 269 24

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

Site-directed mutagenesis of autolysis sites in the human immunodeficiency virus type 1 (HIV-1) protease was applied in an analysis of enzyme specificity; the protease served, therefore, as both enzyme and substrate in this study. Inspection of natural substrates of all retroviral proteases revealed the absence of beta-branched amino acids at the P1 site and of Lys anywhere from P2 through P2'. Accordingly, several mutants of the HIV-1 protease were engineered in which these excluded amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant enzymes were evaluated in terms of their resistance to autodegradation. All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia coli, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites. Some, however, were not viable enzymatically because of poor physical characteristics. This was the case for mutants having Lys replacements of Glu residues at P2' and for another in which all three P1 leucines were replaced by Ile. However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical properties, specificity, and susceptibility to inhibition of the wild-type enzyme. Q7K/L33I/L63I should find useful application as a stable surrogate of the HIV-1 protease. Overall, our results can be interpreted relative to a model in which the active HIV-1 protease dimer is in equilibrium with monomeric, disordered species which serve as the substrates for autolysis.
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PMID:The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties. 806 16

Inhibitors of the human immunodeficiency virus type 1 protease represent a promising class of antiviral drugs for the treatment of AIDS, and several are now in clinical trials. Here, we report the in vitro selection of viral variants with decreased sensitivity to a C2-symmetric protease inhibitor (A-77003). We show that a single amino acid substitution (Arg to Gln or Lys) at position 8 of the protease results in a substantial decrease in the inhibitory activity of the drug on the enzyme and a comparable increase in viral resistance. These findings, when analyzed by using the three-dimensional structure of the protease-drug complex, provide a strategic guide for the future development of inhibitors of the human immunodeficiency virus type 1 protease.
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PMID:Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor. 810 64

Autoproteolysis of the retroviral aspartyl proteases is a major obstacle to purification and analysis of these enzymes. A mutagenic approach to rendering autolytic cleavage sites less labile was applied to the primary cleavage site between Leu5 and Trp6 in human immunodeficiency virus-1 (HIV-1) protease. From predictions based on known substrates it was concluded that amino acids Lys or Ser in place of Gln at position 7 would prevent cleavage at the Leu5-Trp6 peptide bond, therefore stabilizing the protein. Autoproteolytic stability was enhanced at least 100-fold by these mutations. At longer time points the protease was degraded at secondary sites which contained adequate substrate sequences but were conformationally restricted. Conversely, a mutation in HIV-2 protease which changed Lys7 to Gln rendered the protein 3-fold less stable and shifted the position of the initial autoproteolytic cleavage from Phe3-Ser4 to Leu5-Trp6. The effects of these mutations demonstrate that small changes in protein sequence can have a major impact on their autoproteolytic stability. The work described here suggests a general method for stabilizing proteases and perhaps other recombinantly produced proteins to autolysis.
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PMID:Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions. 850 18

To evaluate the available peptidic and pseudopeptidic inhibitors of HIV protease for their possible in vivo activity, a screening test using Escherichia coli was established. E. coli cells carrying the plasmid pET9c-PR containing the gene for HIV-1 protease under the control of a T7-promotor are grown in the absence and in the presence of inhibitors. The action of the toxic protease produced by the cells is counteracted by the inhibitors. Provided sufficient membrane permeability of the inhibitors exists, this results in accelerated cell growth. From the peptides known to be active in an in-vitro enzyme test, most compounds inhibit HIV protease only to a limited degree in this test. However, two short peptides (Ac-Ser-Tyr-Glu-Leu and Lys-Ile-Ser-Tyr-Asp-Tyr) protect cell growth to an considerabe extent, thus indicating that they reach the E. coli cytosol and there block HIV protease. Two pseudopeptides known to be very potent in the enzyme test (SDZ PRI 053 and CIBA 61755) also inhibit HIV-1 protease strongly in this cell growth test.
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PMID:Screening of inhibitors of HIV-1 protease using an Escherichia coli cell assay. 914 91


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