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B M Dunn

Publications and source records attributed to B M Dunn.

At least 19 recordsLinked to original sources
PubMed2001

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.

Binding Sites↗
PubMed2001

The 2-A postdoc.

Explore the source record for details and available documents.

Carboxypeptidases↗
PubMed2001

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.

Adolescent↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

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.

Journal Article↗
PubMed2000

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.

Adult↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

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.

Aspartic Acid Endopeptidases↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Aspartic Acid Endopeptidases↗
PubMed1999

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.

Aspartic Acid Endopeptidases↗
PubMed1998

Prime region subsite specificity characterization of human cathepsin D: the dominant role of position 128.

In order to contribute to our understanding of cathepsin D (CatD) active site specificity, two series of chromogenic octapeptides with systematic substitutions in positions P2' and P3' were synthesized. This panel was characterized with native human liver cathepsin D (nHuCatD) and yielded information concerning specificity trends within the S2' and S3' subsites. The pepstatin inhibited crystal structure of nHuCatD (Baldwin et al., 1993) was then utilized in conjunction with these subsite preference data to identify residues suspected of contributing to "prime" side subsite specificity. These residues were targeted for site-directed mutagenesis using the re-engineered recombinant model, "short" pseudocathepsin D (Beyer & Dunn, 1996). As a result of these analyses it was determined that prime region subsites do contribute to the unique specificity of human CatD. Furthermore, it was ascertained that the poly-proline loop does not have an active role in S3' subsite specificity. Lastly, it appears that Ile128 has a dominant role on S2' subsite specificity whereas Val130 does not.

Binding Sites↗
PubMed1998

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.

Antiviral Agents↗