Proteases, protease inhibitors and protease-derived peptides. Importance in human pathophysiology and therapeutics. Symposium proceedings. Snowmass, Colorado, August 1992.
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Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease.
A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.
Amino Acid Sequence↗
Pro region C-terminus:protease active site interactions are critical in catalyzing the folding of alpha-lytic protease.
alpha-Lytic protease is encoded with a large (166 amino acid) N-terminal pro region that is required transiently both in vivo and in vitro for the correct folding of the protease domain [Silen, J. L. , and Agard, D. A. (1989) Nature 341, 462-464; Baker, D., et al. (1992) Nature 356, 263-265]. The pro region also acts as a potent inhibitor of the mature enzyme [Baker, D., et al. (1992) Proteins: Struct.,Funct., Genet. 12, 339-344]. This inhibition is mediated through direct steric occlusion of the active site by the C-terminal residues of the pro region [Sohl, J. L., et al. (1997) Biochemistry 36, 3894-3904]. Through mutagenesis and structure-function analyses we have begun to investigate the mechanism by which the pro region acts as a single turnover catalyst to facilitate folding of the mature protease. Of central interest has been mapping the interface between the pro region and the protease and identifying interactions critical for stabilizing the rate-limiting folding transition state. Progressive C-terminal deletions of the pro region were found to have drastic effects on the rate at which the pro region folds the protease but surprisingly little effect on inhibition of protease activity. The observed kinetic data strongly support a model in which the detailed interactions between the pro region C-terminus and the protease are remarkably similar to those of known substrate/inhibitor complexes. Further, mutation of two protease residues near the active site have significant effects on stabilization of the folding transition state (kcat) or in binding to the folding intermediate (KM). Our results suggest a model for the alpha-lytic protease pro region-mediated folding reaction that may be generally applicable to other pro region-dependent folding reactions.
Amino Acid Sequence↗
The alpha-lytic protease pro-region does not require a physical linkage to activate the protease domain in vivo.
alpha-Lytic protease, an extracellular serine protease of Lysobacter enzymogenes 495, is synthesized as a pre-pro-protein. Previously it has been shown that when expressed in Escherichia coli, the protein is autocatalytically processed in the periplasmic space, and that the functional protease domain accumulates extracellularly. Engineered proteins lacking the 166 amino-acid pro-region were enzymatically inactive and remained cell-associated. By independently expressing the pro- and protease domains in vivo, evidence is provided here that direct covalent linkage is not required for production of active protease. We postulate that the pro-region acts as a template to promote the folding of the protease domain into an active configuration. Our results, combined with recent experiments on the evolutionarily unrelated subtilisin E (ref. 3), suggest that the ability of the pro-region of these bacterial proteases to facilitate folding of their protease domains is not a curiosity of a single system, but may reflect a general property of extracellular bacterial serine proteases.
Alkaline Phosphatase↗
Upregulation of neurotoxic serine proteases, prothrombin, and protease-activated receptor 1 early after spinal cord injury.
Apoptosis, well-established in development and now also in degenerative disease, occurs with regularity in several cell compartments early after controlled contusion spinal cord injury (SCI). Cell death in astrocytic, microglial, and neuronal populations peaks at 3 days, while oligodendroglial apoptosis is found 10-14 days later. In this regard, the executioners of apoptosis, the caspase proteases, are also activated within 3 days of SCI. On the other hand, serine proteases, which have been shown to initiate apoptosis and activate caspases in culture models, have not been extensively studied in regards to nervous system trauma. As part of an ongoing effort to examine the spectrum of genes that are up- and downregulated in the injured rat spinal cord, we synthesized serine protease family specific primers to take advantage of conserved residues in the charge relay system and the codon preferences of these mammalian genes. These primers were then employed in a modified, family-specific differential mRNA display technique. One specific serine protease gene we found that was upregulated after injury was prothrombin. Qualitative and quantitative RT-PCR techniques indicated that this increase occurred early, already evident at 8 h after injury, and reached a maximum level fourfold above baseline at 24 h. Peak expression for prothrombin mRNA occurred prior to peak levels of apoptosis in astrocytic, microglial and neuronal compartments at 72 h. Of additional interest, gene database mining revealed that prothrombin shared approximately 48% similarity with myelencephalon-specific protease (MSP), a neurotoxic serine protease previously found to be increased two- to threefold at 3 days after excitotoxic SCI. Since thrombin induces apoptosis in murine and chick motor and rat hippocampal neurons by activating a member of the novel protease-activated receptor (PAR) gene family known as PAR-1, we also analyzed PAR-1 by similar techniques and found that it, too, was upregulated after SCI with the same kinetics as prothrombin. We confirmed these results with gene array analyses that revealed more than one trypsin subfamily serine protease was activated by SCI. They imply the possibility of using specific, tissue-directed serine protease inhibition at translational or transcriptional levels, and offer a potential paradigm shift in drug discovery for SCI to limit the extent of apoptosis, and consequent functional loss, in the human spinal cord.
Amino Acid Motifs↗
IgA protease produced by Streptococcus sanguis and antibody production against IgA protease in patients with Behçet's disease.
The production of IgA protease in twelve strains of Streptococcus sanguis isolated from patients with Behcet's disease (BD) was examined. Protease activity was detected in 10 out of 12 strains. The protease was purified from one representative strain, S. sanguis 113-20, by employing Rotofor and DEAE-Sephacel chromatography. The molecular mass of the purified protease was approximately 100 kDa, and it cleaved the proline-threonine site of the IgA. Both IgG and IgA titers against the cells (113-20) and the purified IgA protease in the sera of BD patients and healthy controls, 36 each, were assayed. The IgG titers against the cells and protease were not significant in the BD patients or controls, but the IgA titers against the cells and protease in the BD patients were significantly higher than those of the controls. These data indicate that the BD patients are infected with IgA protease-producing S. sanguis strains, which cause an increase of IgA titer against these organisms and IgA protease antigen. Since the organisms can proliferate in BD patients for a long period of time (years), it seems that IgA antibodies cannot effectively eliminate the organisms.
Amino Acid Sequence↗
Differential activation of two protease-activated protein kinases from reticulocytes by a Ca2+-stimulated protease and identification of phosphorylated translational components.
Two protein kinases have been partially purified from rabbit reticulocytes and shown to be activated by limited proteolysis with trypsin [S.M. Tahara and J.A. Traugh (1981) J. Biol. Chem. 256, 11558-11564; P.T. Tuazon, W.C. Merrick, and J.A. Traugh (1980) J. Biol. Chem. 255, 10954-10958]. Reticulocyte lysate was examined for protease activities which might be involved in activation of the protein kinases in vivo. Two neutral proteases, differentially activated by Fe2+ and Ca2+, were identified and partially purified. The Ca2+-stimulated protease specifically activated protease-activated kinase II; no effect was observed on protease-activated kinase I. The Fe2+-stimulated protease was not active on either protein kinase. The protease-activated kinases were examined using initiation factors (eIF) and 40-S ribosomal subunits as substrate. Protease-activated kinase I phosphorylated one subunit of eIF-3 (Mr 130000), eIF-4B and 40-S ribosomal protein S10. Protease-activated kinase II modified the beta subunit of eIF-2 (Mr 53000) and 40-S ribosomal protein S6. The substrate specificities are unique when compared with other cAMP-dependent and cAMP-independent protein kinases from reticulocytes.
Calcium↗
Regulation of exocellular proteases in Neurospora crassa: role of Neurospora proteases in induction.
Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.
Chromatography↗
Competition between elastase and related proteases from human neutrophil for binding to alpha1-protease inhibitor.
The protease-antiprotease imbalance that is characteristic of most inflammatory lung disorders depends on the spatial-temporal regulation of active inhibitor and protease concentrations in lung secretions. We have studied the competition between the three main serine proteases from human neutrophil primary granules in their binding to alpha1-Pi, the main serine proteases inhibitor in lung secretions. Elastase was the only target of alpha1-Pi when identical molar amounts of purified inhibitor and the three proteases were tested together. The other two proteases were only inhibited once elastase was saturated. Elastase remained the preferred target of inhibitors when bronchoalveolar lavage fluids from patients with lung pneumonia and acute respiratory distress syndrome were used as the source of inhibitors, in spite of the presence of additional inhibitors in lung secretions. Since neutrophil proteases are expressed at the neutrophil surface, we also measured residual activities of membrane-bound proteases after purified neutrophils were incubated with bronchoalveolar fluids. Again, elastase was the preferred target of the inhibitors. We conclude that protease 3 and cathepsin G are not controlled as efficiently as elastase in lung secretions, a feature that must be taken into account when developing inhibitor-based anti-inflammatory therapies.
Binding, Competitive↗
Studies on the role of human insulin-like growth factor-II (IGF-II)-dependent IGF binding protein (hIGFBP)-4 protease in human osteoblasts using protease-resistant IGFBP-4 analogs.
To characterize the insulin-like growth factor binding protein-4 (IGFBP-4) protease produced by human osteoblasts (hOBs), we localized and determined the role of the proteolytic domains in human IGFBP-4 (hIGFBP-4) in modulating IGF-II actions. N-terminal amino acid sequence and mass spectrometric analyses of the 6xHis-tagged IGFBP-4 proteolytic fragments revealed that Met135-Lys136 was the only cleavage site recognized by the IGF-II-dependent IGFBP-4 protease produced by hOBs. This cleavage site was confirmed by the finding that deletion of His121 to Pro141 blocked proteolysis. However, unexpectedly, deletion of Pro94 to Gln119 containing no cleavage site had no effect on IGF-II binding activity but blocked proteolysis. Addition of the synthetic peptide corresponding to this region at concentrations of 250 or 1000 molar excess failed to block IGFBP-4 proteolysis. These data suggest that residues 94-119 may be involved in maintaining the IGFBP-4 conformation required to expose the cleavage site rather than being involved in direct protease-substrate binding. To determine the physiological significance of the IGF-II-dependent IGFBP-4 protease, we compared the effect of the wild-type IGFBP-4 and the protease-resistant IGFBP-4 analogs in blocking IGF-II-induced cell proliferation in normal hOBs, which produce IGFBP-4 protease, and MG63 cells, which do not produce IGFBP-4 protease. It was found that protease-resistant IGFBP-4 analogs were more potent than the wild-type protein in inhibiting IGF-II-induced cell proliferation in hOBs but not in MG63 cells. These data suggest that IGFBP-4 proteolytic fragments are not biologically active and that IGFBP-4 protease plays an important role in regulating IGFBP-4 bioavailability and consequently the mitogenic activity of IGFs in hOBs.
Amino Acid Sequence↗
Analysis of in situ protease activity in chronic adult periodontitis patients: expression of activated MMP-2 and a 40 kDa serine protease.
BACKGROUND: Periodontitis is characterized by extensive destruction of the gingival tissues and associated supporting structures of the teeth. Although the pathogenesis of the various forms of this disease is not completely understood, host-derived proteases are believed to have an important role. In this study, we analyzed human tissue samples from chronic adult periodontitis patients to assess the levels of specific proteases and determine the effect of pH and tetracyclines on their activity. METHODS: Gingival tissue samples were obtained from patients with chronic adult periodontitis (probing depths ranged from 5 to 9 mm) and periodontally healthy controls. Tissue extracts were prepared and analyzed for protease activity by zymography and Western blotting. RESULTS: Maximal protease activity from clinically normal and diseased tissues was observed at pH 8. Latent matrix metalloproteinase (MMP)-9 and MMP-2 were expressed in all samples examined, while active MMP-2 was detected only in tissues obtained from patients with clinical disease. The MMP activities were differentially inhibited by derivatives of tetracycline. At pH 6, a protease with a mass of approximately 40 kDa was observed in diseased samples. The enzymatic activity was inhibited by phenylmethylsulfonyl fluoride, suggesting it is a serine protease. CONCLUSIONS: The results of the current study substantiate the proposed role of host-derived proteases in the pathogenesis of chronic adult periodontitis. Specifically, they indicate that activated MMP-2 and a 40 kDa serine protease are involved in tissue destruction associated with this form of periodontal disease and also suggest that tissue pH influences protease activity in situ.
Adult↗
A chymotrypsin-type serine protease in rat basophilic leukemia cells: evidence for its immunologic identity with atypical mast cell protease.
Activity of a chymotrypsin-type serine protease was found in a subline of rat basophilic leukemia (RBL-2H3) cells. The protease was immunologically cross-reactive with anti-atypical mast cell protease immunoglobulin (Ig) G, and its activity was inhibited in a dose-dependent manner by the antibody. The apparent m.w. of the protease that reacted with the antibody was 25,000, which was identical with that of atypical mast cell protease in rat mucosal mast cells. These results show that the chymotrypsin type serine protease in RBL-2H3 cells is immunologically identical with atypical mast cell protease, which was first purified from rat small intestine. Immunohistochemical studies showed that the protease was located not only in intracytoplasmic granules but also in organelles synthesizing protein, such as cisternae of the rough endoplasmic reticulum, perinuclear spaces, and the Golgi apparatus. However, no immunoreactivity was demonstrated in rat basophils. The activity of the protease increased in the exponential phase of growth of RBL-2H3 cells in which some activity was also detected in the medium, and it decreased in the late stationary phase.
Animals↗
Studies on protease from marine bacteria. 1 Isolation of marine Pseudomonas sp 145-2 and purification of protease.
Marine Pseudomonas strain 145-2 having the ability to produce extracellular protease was isolated from Uwa sea water, Ehime prefecture in Japan. This strain grows at 0 degrees - 37 degrees C, but protease production was observed only during 6-10 h cultivation at 20 degrees - 30 degrees C, when casein, as the nitrogen and carbon source in a concentration of 0.5-1.0%, was used. Maximum production of protease by the strain was also observed. Na, K, Ca and Mg ions were needed for growth and production of protease. Two extracellular proteases (proteases I and II) were purified from the culture supernatant by ammonium sulphate fractionation following Sephadex gel chromatography. Protease I had a larger molecular weight than protease II. After purification, proteases I and II were revealed as homogeneous by acrylamide gel electrophoresis and immunoelectrophoresis.
Amino Acids↗
Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro.
Thermally aggregated, endogenous proteins of Escherichia coli form a distinct fraction, denoted S, which is separable by sucrose-density-gradient centrifugation. It was shown earlier that DnaK, DnaJ, IbpA and IbpB heat-shock proteins are associated with the S fraction. Comparison of the rise and decay of the S fraction in mutants defective for heat-shock proteases Lon (La), Clp, HtrA (DegP, Do) and in wild-type strains made studies of proteolysis and the function of the heat-shock response possible in vivo. Different timing and the extent of action of particular proteases was revealed by the initial size and decay kinetics of the S fraction. The proteases Lon, Clp, and HtrA all participated in removal of the aggregated proteins. Mutation in the gene encoding ClpB caused the most prominent effect (47% stabilization of the S fraction). The correlation between the disappearance of the S fraction and proteolytic activity was supported by the result of the in vitro reaction. Approximately one third of the isolated S fraction was converted to trichloroacetic acid-soluble products by the purified HtrA protease. Mg2+ ions stimulated the reaction, in contrast to the reaction of the HtrA protease with casein. The digestion of the aggregated proteins, unlike the digestion of casein, by HtrA protease in vitro was inhibited by added DnaJ, which might reflect protection of the aggregated proteins in vivo by DnaJ from excessive degradation. One might expect that such an activity of DnaJ would promote denatured protein renaturation versus proteolysis. Moreover, among the aggregated proteins that are discernible by electrophoresis, none could be identified as being more susceptible than any other to HtrA degradation. The separation pattern of these proteins before and after the in vitro digestion did not show a difference corresponding to the loss of about 30% of constituting proteins. This was interpreted as recognition by the HtrA protease of a state of protein denaturation rather than specific amino acid sequences in particular proteins. We conclude that the fraction consisting of proteins heat-aggregated in vivo (i.e. the S fraction) contains endogenous substrates for the heat-shock proteases tested. Their use for in vitro reaction reveals information that is in some respects different from that obtained with exogenous substrates such as casein.
ATP-Dependent Proteases↗
Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La.
The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. We have partially purified another ATP-dependent protease from lon-cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [3H]methyl-casein to acid-soluble products in the presence of ATP and Mg2+. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. We propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show proteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. [Katayama-Fujimura, Y., Gottesman, S. & Maurizi, M. R. (1987) J. Biol. Chem. 262, 4477-4485] indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.
ATP-Dependent Proteases↗
Resistance-related mutations in the HIV-1 protease gene of patients treated for 1 year with the protease inhibitor ritonavir (ABT-538).
OBJECTIVE: To define genotypic and phenotypic resistance patterns following prolonged therapy with the protease inhibitor ritonavir (ABT-538). DESIGN: Seven HIV-1-infected patients, all but one previously treated with dideoxynucleoside analogues (zidovudine, didanosine, zalcitabine), were treated for 1 year with ritonavir. METHODS: Direct solid-phase sequencing of the protease gene starting from plasma derived viral RNA followed by comparison to phenotypic drug resistance data. RESULTS: The most frequent amino-acid substitutions occurring upon administration of the protease inhibitor were V82A/F (substrate binding site), I54V (flap region), A71V and L10I. Additional mutations found in more than one patient were I15V, M36I, I84V and I93L. Mutation L63P was found both in pre- and post-ritonavir samples. Phenotypic drug resistance assays confirmed resistance to ritonavir in post-treatment samples (approximately 170-fold) and showed cross-resistance to indinavir (approximately 30-fold) and partially to saquinavir (approximately fivefold). At 1 year of treatment, one patient without known resistance-associated mutations in the protease gene still showed a substantial rise in CD4 cell count accompanied by a more than 2.4 log decrease in RNA viral load. However, at week 78, mutations R8Q, E34K, R57K, L63P and I84V were detected and the treatment benefit was partially lost. CONCLUSIONS: Long-term treatment with ritonavir is associated with the emergence of multiple mutations in the HIV-1 protease gene. The mutations L10I, I54V, L63P, A71V, V82A/F and I84V correspond to known drug-resistance mutations for ritonavir and other protease inhibitors. Phenotypic resistance to ritonavir was detected in a majority of ritonavir-treated patients at 1 year of treatment. In addition, long-term ritonavir treatment selects for cross-resistance to the protease inhibitors indinavir and saquinavir. This argues against sequential therapy with several protease inhibitors. Delayed resistance in one patient was accompanied with a prolonged increase in CD4 cell count and decrease in viral load suggesting a temporary benefit of treatment.
Amino Acid Sequence↗
An alternate binding site for the P1-P3 group of a class of potent HIV-1 protease inhibitors as a result of concerted structural change in the 80s loop of the protease.
Structures of the complexes of HIV protease inhibitor L--756,423 with the HIV-1 wild-type protease and of the inhibitors Indinavir, L-739,622 and Saquinavir with the mutant protease (9X) containing nine point mutations (Leu10Val, Lys20Met, Leu24Ile, Ser37Asp, Met46Ile, Ile54Val, Leu63Pro, Ala71Val, Val82Thr) have been determined. Comparative analysis of these structures reveals an alternate binding pocket for the P1-P3 group of Indinavir and L--756, 423. The alternate binding pocket is a result of concerted structural change in the 80s loop (residues 79-82) of the protease. The 80s loop is pulled away from the active site in order to accommodate the P1-P3 group, which is sandwiched between the flap and the 80s loop. This structural change is observed for the complexes of the wild type as well as the 9X mutant protease. The study reveals that the 80s loop is an intrinsically flexible loop in the wild-type HIV-1 protease and that mutations in this loop are not necessary to result in conformational changes. Conformation of this loop in the complex depends primarily upon the nature of the bound inhibitor and may be influenced by mutations in the protease. The results underscore the need to understand the intrinsic structural plasticity of the protease for the design of effective inhibitors against the wild-type and drug-resistant enzyme forms. In addition, the alternate binding pocket for the P1-P3 group of Indinavir and L--756,423 may be exploited for the design of potent inhibitors.
Amino Acid Substitution↗
Crystal structure at 1.9-A resolution of human immunodeficiency virus (HIV) II protease complexed with L-735,524, an orally bioavailable inhibitor of the HIV proteases.
L-735,524 is a potent, orally bioavailable inhibitor of human immunodeficiency virus (HIV) protease currently in a Phase II clinical trial. We report here the three-dimensional structure of L-735,524 complexed to HIV-2 protease at 1.9-A resolution, as well as the structure of the native HIV-2 protease at 2.5-A resolution. The structure of HIV-2 protease is found to be essentially identical to that of HIV-1 protease. In the crystal lattice of the HIV-2 protease complexed with L-735,524, the inhibitor is chelated to the active site of the homodimeric enzyme in one orientation. This feature allows an unambiguous assignment of protein-ligand interactions from the electron density map. Both Fourier and difference Fourier maps reveal clearly the closure of the flap domains of the protease upon L-735,524 binding. Specific interactions between the enzyme and the inhibitor include the hydroxy group of the hydroxyaminopentane amide moiety of L-735,524 ligating to the carboxyl groups of the essential Asp-25 and Asp-25' enzymic residues and the amide oxygens of the inhibitor hydrogen bonding to the backbone amide nitrogen of Ile-50 and Ile-50' via an intervening water molecule. A second bridging water molecule is found between the amide nitrogen N2 of L-735,524 and the carboxyl oxygen of Asp-29'. Although other hydrogen bonds also add to binding, an equally significant contribution to affinity arises from hydrophobic interactions between the protease and the inhibitor throughout the pseudo-symmetric S1/S1', S2/S2', and S3/S3' regions of the enzyme. Except for its pyridine ring, all lipophilic moieties (t-butyl, indanyl, benzyl, and piperidyl) of L-735,524 are rigidly defined in the active site.
Aspartic Acid Endopeptidases↗