Inhibitor complexes of the Pseudomonas serine-carboxyl proteinase.

Crystal structures of the serine-carboxyl proteinase from Pseudomonas sp. 101 (PSCP), complexed with a number of inhibitors, have been solved and refined at high- to atomic-level resolution. All of these inhibitors (tyrostatin, pseudo-tyrostatin, AcIPF, AcIAF, and chymostatin, as well as previously studied iodotyrostatin and pseudo-iodotyrostatin) make covalent bonds to the active site Ser287 through their aldehyde moieties, while their side chains occupy subsites S1-S4 of the enzyme. The mode of binding of the inhibitors is almost identical for their P1 and P2 side chains, while significant differences are observed for P3 and P4 (if present). Kinetic parameters for the binding of these nanomolar inhibitors to PSCP have been established and correlated with the observed mode of binding. The preferences of this enzyme for a larger side chain in P2 as well as Tyr or Phe in P1 are explained by the size, shape, and characteristics of the S2 and S1 regions of the protein structure, respectively. Networks of hydrogen bonds involving glutamic and aspartic acids have been analyzed for the atomic-resolution structure of the native enzyme. PSCP contains a calcium-binding site that consists of Asp328, Asp348, three amide carbonyl groups, and a water molecule, in almost perfect octahedral coordination. The presence of Ca(2+) cation is necessary for the activity of the enzyme.

Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes.

The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions. Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0-1.4 A resolution. The structure of PSCP comprises a single compact domain with a diameter of approximately 55 A, consisting of a seven-stranded parallel beta-sheet flanked on both sides by a number of helices. The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue. Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287.

Human immunodeficiency virus type 1 protease genotype predicts immune and viral responses to combination therapy with protease inhibitors (PIs) in PI-naive patients.

Protease genotype, as a variable in outcome to combination therapy for human immunodeficiency virus (HIV) type 1 infection, was evaluated among protease inhibitor-naive children and adolescents who had received extensive treatment with reverse-transcriptase inhibitors. After 24 weeks of combination therapy, 35% had viral and immune success (VSIS patients), 19% had viral and immune failure (VFIF patients), and 46% had viral failure but marked improvement in CD4 T cells (VFIS patients). Disease stage was the only pretherapy clinical variable associated with outcome (P=.02). Although reverse-transcriptase genotype was unrelated to outcome, pretherapy protease genotype was related significantly to therapy response (P=.005). Odds for immune or viral failure were 17.7 to 1 and 2.5 to 1, respectively, for protease genotype as a single variable. Protease genotype combined with disease stage and CD4 cell percentage predicted correct therapy response for 81% of patients (100% of VFIF, 78% of VSIS, and 75% of VFIS patiens). Naturally occurring amino acid polymorphisms in protease provide sensitive biomarkers for treatment response among inhibitor-naive patients with advanced HIV disease.

The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae.

The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.

Overview of pepsin-like aspartic peptidases.

The aspartic peptidase family of enzymes has been implicated in a variety of disease states, from stomach ulcers, to breast cancer, and even Alzheimer's Disease. This unit describes the major characteristics of the aspartic peptidases, including mechanism of action, subcellular and tissue localization, and biological substrate specificity.

Subsite preferences of pepstatin-insensitive carboxyl proteinases from prokaryotes: kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase.

Kumamolysin, a carboxyl proteinase from Bacillus novosp. MN-32, is characterized by its thermostability and insensitivity to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidated using two series of synthetic chromogenic substrates: P(5)-P(4)-P(3)-P(2)-Phe*Nph (p-nitrophenylalanine: *cleavage site)-P(2)'-P(3)', in which the amino acid residues at the P(5)-P(2), P(2)' and P(3)' positions were systematically substituted. Among 74 substrates, kumamolysin was shown to hydrolyze Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this peptide were K(m) = 41+/-5 microM, k(cat) = 176+/- 10 s(-1), and k(cat)/K(m) = 4.3+/-0.6 mM(-1) x s(-1). These systematic analyses revealed the following features: (i) Kumamolysin had a unique preference for the P(2) position. Kumamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P(2) position; this was also observed for the pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al. (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomonas sp. 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas sp. T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P(2) position. (ii) Kumamolysin preferred such charged amino acid residues as Glu or Arg at the P(2)' position, suggesting that the S(2)' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4. In general, the S(2)' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydrophilic nature of the S(2)' subsite was confirmed to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases from prokaryotes.

Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3.

The three-dimensional structures of pepsin inhibitor-3 (PI-3) from Ascaris suum and of the complex between PI-3 and porcine pepsin at 1. 75 A and 2.45 A resolution, respectively, have revealed the mechanism of aspartic protease inhibition by this unique inhibitor. PI-3 has a new fold consisting of two domains, each comprising an antiparallel beta-sheet flanked by an alpha-helix. In the enzyme-inhibitor complex, the N-terminal beta-strand of PI-3 pairs with one strand of the 'active site flap' (residues 70-82) of pepsin, thus forming an eight-stranded beta-sheet that spans the two proteins. PI-3 has a novel mode of inhibition, using its N-terminal residues to occupy and therefore block the first three binding pockets in pepsin for substrate residues C-terminal to the scissile bond (S1'-S3'). The molecular structure of the pepsin-PI-3 complex suggests new avenues for the rational design of proteinaceous aspartic proteinase inhibitors.

Methemoglobinemia after topical anesthesia with lidocaine and benzocaine for a difficult intubation.

Methemoglobinemia is an uncommon cause of cyanosis; however, rapid diagnosis is critical to avoid potentially fatal consequences. Several local anesthetics can precipitate methemoglobinemia in susceptible patients. This case report of acute methemoglobinemia occurred during fiberoptic intubation in an awake patient without a past medical history of adverse reactions to local anesthetics.

The two sides of enzyme-substrate specificity: lessons from the aspartic proteinases.

Like most proteolytic enzymes, the aspartic proteinases bind substrates and most inhibitors within an extended active site cleft. Bound ligands typically adopt a beta-strand conformation. Interactions with groups on both sides of the cleft determine the primary as well as secondary specificity of the enzymes. We have pursued the discovery of the sometimes subtle distinctions between members of the aspartic proteinase family by two routes. In the first case, we have constructed sets of oligopeptide substrates with systematic variation in each position to assess interactions at one position at a time. In the second type of experiment, we have altered residues of the enzymes in order to test theories of selectivity. The combination of the two approaches has provided a better understanding of the forces involved in determining specificity of enzyme action.

The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix.

Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.2 and 1.8 A, respectively, for complexes of proteinase A with full-length IA3 and with a truncated form consisting only of residues 2-34, reveal an unprecedented mode of inhibitor-enzyme interactions. Neither form of the free inhibitor has detectable intrinsic secondary structure in solution. However, upon contact with the enzyme, residues 2-32 become ordered and adopt a near-perfect alpha-helical conformation. Thus, the proteinase acts as a folding template, stabilizing the helical conformation in the inhibitor, which results in the potent and specific blockage of the proteolytic activity.

Chimeric aspartic proteinases and active site binding.

Two chimeric enzymes were constructed by exchanging domains between porcine pepsinogen and rhizopuspepsinogen in order to examine the contributions of the subsites present on different domains toward enzymatic specificity. Both chimeras exhibited the characteristic features of aspartic proteinases, such as auto-activation at low pH and abrogation of enzymatic activity by pepstatin. The activity of the chimera containing the N-terminal domain of rhizopuspepsinogen and the C-terminal domain of porcine pepsinogen (rhzNppC) could be observed by HPLC after prolonged incubation with the substrates. In contrast, the reciprocal chimera, ppNrhzC, containing the N-terminal domain of porcine pepsinogen and the C-terminal domain of rhizopuspepsinogen exhibited catalytic activity, measurable by a spectrophotometric assay. Kinetic data and inhibitor analyses strongly suggest that interdependency may exist between adjacent subsites contributed by different domains. Therefore, in order to develop an optimal substrate or inhibitor, the effect of adjacent residues of the ligand has to be examined along with the preferences for each subsite.

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.

Comparison of inhibitor binding to feline and human immunodeficiency virus proteases: structure-based drug design and the resistance problem.

The design and synthesis of compounds targeted against human immunodeficiency virus 1 (HIV-1) protease have resulted in effective antiviral therapies. However, the rapid replication of the virus and the inherent mutability of the viral genome result in the outgrowth of resistant strains in the majority of patients. Thus, there is a continuing need to develop new antiprotease compounds that may bind more effectively to the resistant forms of protease. This contribution examines the binding of a single inhibitor to two different retroviral proteases, HIV-1 protease and feline immunodeficiency virus protease. Despite the overall similarity of the related retroviral enzymes, specific substitutions within the binding site cavity provide a distinctly different binding landscape that dramatically alters the affinity of compounds. Through this comparison, insights have been obtained into new strategies for drug design. New compounds based on these concepts have been tested against the two enzymes.

Subsite preferences of pepstatin-insensitive carboxyl proteinases from bacteria.

Pseudomonas sp. 101 carboxyl proteinase (PCP) and Xanthomonas sp. T-22 carboxyl proteinase (XCP), the first and second unique carboxyl proteinases from prokaryotes to be isolated and characterized, are not inhibited by the classical carboxyl proteinase inhibitor pepstatin. In this study, we elucidated their subsite preferences by using a series of synthetic chromogenic substrates, Lys-Pro-Ile(P3)-Glu(P2)-Phe*Nph-Arg(P2')-Leu(P3') (Nph is p-nitrophenylalanine, Phe*Nph is the cleavage site) with systematic substitutions at the P3, P2, P2', and P3' positions. Among 45 substrates tested, the best substrate for PCP had a Leu replacement at the P2 position (kcat = 27.2 s-1, Km = 4.22 microM, kcat/Km = 6.43 microM-1.s-1), and that for XCP had an Ala replacement at the P3 position (kcat = 79.4 s-1, Km = 6.05 microM, kcat/Km = 13.1 microM-1. s-1). PCP and XCP preferred such charged amino acid residues as Glu, Asp, Arg, or Lys at the P2' position. This suggested that the S2' subsites of PCP and XCP are occupied by hydrophilic residues, similar to that of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [Shibata et al. (1998) J. Biochem. 124, 642-647]. In contrast, the S2' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydophilic nature of the S2' subsite appears to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases.

Toward a universal inhibitor of retroviral proteases: comparative analysis of the interactions of LP-130 complexed with proteases from HIV-1, FIV, and EIAV.

One of the major problems encountered in antiviral therapy against AIDS is the emergence of viral variants that exhibit drug resistance. The sequences of proteases (PRs) from related retroviruses sometimes include, at structurally equivalent positions, amino acids identical to those found in drug-resistant forms of HIV-1 PR. The statine-based inhibitor LP-130 was found to be a universal, nanomolar-range inhibitor against all tested retroviral PRs. We solved the crystal structures of LP-130 in complex with retroviral PRs from HIV-1, feline immunodeficiency virus, and equine infectious anemia virus and compared the structures to determine the differences in the interactions between the inhibitor and the active-site residues of the enzymes. This comparison shows an extraordinary similarity in the binding modes of the inhibitor molecules. The only exceptions are the different conformations of naphthylalanine side chains at the P3/P3' positions, which might be responsible for the variation in the Ki values. These findings indicate that successful inhibition of different retroviral PRs by LP-130 is achieved because this compound can be accommodated without serious conformational differences, despite the variations in the type of residues forming the active-site region. Although strong, specific interactions between the ligand and the enzyme might improve the potency of the inhibitor, the absence of such interactions seems to favor the universality of the compound. Hence, the ability of potential anti-AIDS drugs to inhibit multiple retroviral PRs might indicate their likelihood of not eliciting drug resistance. These studies may also contribute to the development of a small-animal model for preclinical testing of antiviral compounds.

Substrate specificity of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4.

Bacillus coagulans J-4 carboxyl proteinase, designated as J-4, is characterized as alcohol-resistant and insensitive to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucinemetylester, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Here, its substrate specificity was elucidated by using two series of chromogenic substrates, Lys-Pro-Ala-Lys-Phe*Nph (p-nitrophenylalanine:* is cleavage site)-Arg-Leu (XVI) and Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu (RS6), in which the amino acid residues at positions P5-P2, P2', and P3' were systematically substituted. Kinetic parameters were determined for both sets of peptides. J-4 was shown to hydrolyze Lys-Pro-Ala-Ala-Phe-Nph-Arg-Leu most effectively among the XVI series. The kinetic parameters of this peptide were Km = 20.0 +/- 3.24 microM, kcat = 15.4 +/- 0.71 s-1, and kcat/Km = 0.769 +/- 0.128 microM-1.s-1. Among the RS6 series, Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu was hydrolyzed most effectively. The kinetic parameters of this peptide were Km = 13.7 +/- 1.30 microM, kcat = 9.65 +/- 0.38 s-1, and kcat/Km = 0.704 +/- 0.072 microM-1.s-1. These systematic analyses revealed that J-4 had a unique preference for the P2 position: J-4 preferentially hydrolyzed peptides having an Ala or Pro residue in the P2 position. Other carboxyl proteinases preferred peptides having hydrophobic and bulky amino acid residue such as Leu in the P2 position. Thus, J-4 was found to differ considerably in substrate specificity from the other carboxyl proteinases reported so far.

Substrate specificities and kinetic properties of proteinase A from the yeast Saccharomyces cerevisiae and the development of a novel substrate.

The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-1.s-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A.