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B M Dunn

Publications and source records attributed to B M Dunn.

At least 19 recordsLinked to original sources
PubMed1992

Intrinsic activity of precursor forms of HIV-1 proteinase.

The wild-type -Phe*Pro- bond located at the N-terminus of the mature aspartic proteinase of HIV-1 was replaced by -Ile-Pro- or -Val-Pro-. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV-proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31-8959, was assessed. A hitherto unobserved cleavage junction (at approximately Ala-Phe*Leu-Gln approximately) in the frame-shift region of the gag-pol viral genome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self-processing at neural pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31-8959.

Amino Acid Sequence↗
PubMed1992

X-ray crystallographic analysis of inhibition of endothiapepsin by cyclohexyl renin inhibitors.

The crystal structures of endothiapepsin, a fungal aspartic proteinase (EC 3.4.23.6), cocrystallized with two oligopeptide renin inhibitors, PD125967 and PD125754, have been determined at 2.0-A resolution and refined to R-factors of 0.143 and 0.153, respectively. These inhibitors, which are of the hydroxyethylene and statine types, respectively, possess a cyclohexylalanine side chain at P1 and have interesting functionalities at the P3 position which, until now, have not been subjected to crystallographic analysis. PD125967 has a bis(1-naphthylmethyl)acetyl residue at P3, and PD125754 possesses a hydroxyethylene analogue of the P3-P2 peptide bond for proteolytic stability. The structures reveal that the S3 pocket accommodates one naphthyl ring with conformational changes of the Asp 77 and Asp 114 side chains, the other naphthyl group residing in the S4 region. The P3-P2 hydroxyethylene analogue of PD125754 forms a hydrogen bond with the NH of Thr 219, thereby making the same interaction with the enzyme as the equivalent peptide groups of all inhibitors studied so far. The absence of side chains at the P2 and P1' positions of this inhibitor allows water molecules to occupy the respective pockets in the complex. The relative potencies of PD125967 and PD125754 for endothiapepsin are consistent with the changes in solvent-accessible area which take place on inhibitor binding.

Amino Acid Sequence↗
PubMed1992

Hepatitis A virus 3C proteinase substrate specificity.

Hepatitis A virus (HAV) 3C proteinase is responsible for processing the viral precursor polyprotein into mature proteins. The substrate specificity of recombinant hepatitis A 3C proteinase was investigated using a series of synthetic peptides representing putative polyprotein junction sequences. Two peptides, corresponding to the viral polyprotein 2B/2C and 2C/3A junctions, were determined to be cleaved most efficiently by the viral 3C proteinase. The kcat/Km values determined for the hydrolysis of a further series of 2B/2C peptides, in which C-terminal and N-terminal amino acids were systematically removed, revealed that P4 through P2' amino acids were necessary for efficient substrate cleavage. The substitution of Ala for amino acids in P1 and P4 positions decreased the rate of peptide hydrolysis by 100- and 10-fold, respectively, indicating that the side chains of Gln in P1 and Leu in P4 are important determinants of substrate specificity. Rates of hydrolysis measured for other P1- and P4-substituted peptides indicate that S1 is very specific for the Gln side chain whereas S4 requires only that the amino acid in P4 be hydrophobic. A continuous fluorescence quench assay was developed, allowing the determination of kcat/Km dependence on pH. The pH rate profile suggests that catalyzed peptide hydrolysis is dependent on deprotonation of a reactive group having a pKa of 6.2 (+/- 0.2). The results of tests with several proteinase inhibitors indicate that this cysteine proteinase, like other picornaviral 3C proteinases, is not a member of the papain family.

3C Viral Proteases↗
PubMed1992

Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites.

The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic- type produces a peptide cleaved very efficiently (kcat greater than 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenylalanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Substrate specificity and kinetic properties of pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. No. 101.

The substrate specificity of the pepstatin-insensitive carboxyl proteinase isolated from Pseudomonas sp. No. 101 was studied by using a series of synthetic chromogenic substrates with general structure P5-P4-P3-P2-P1 *(NO2)Phe-Arg-Leu (P5, P4, P3, P2, P1: a variety of amino acids, (NO2)Phe is p-nitro-L-phenylalanine). The nature of the residues occupying the P2, P3 and P4 positions as well as P1 position had strong influences on kinetic parameters. Among those tested, Lys-Pro-Ile-Glu-Phe*(NO2)Phe-Arg-Leu was the best substrate (Km = 3 microM, kcat = 6.9 s-1, kcat/Km = 2300 mM-1 s-1). The S2 subsite of the enzyme was found to contain one or more basic amino acids while the S4 subsite probably includes one or more acidic amino acids. The pH-dependence of the hydrolysis of Ser-Pro-Ala-Lys-Phe*(NO2)Phe-Arg-Leu was studied. The pK1 and pK2 values for enzyme-substrate complex were found to be 2.97 and 4.92, respectively. Coupled with other results, it seems likely that two active carboxyl residues are involved in the catalytic action of the enzyme. In addition, it was found that a specific peptide inhibitor of the enzyme, tyrostatin, is a compeptive inhibitor with a ki value of 2.6 nM.

Amino Acid Sequence↗
PubMed1992

Inhibition of aspartic proteinases by synthetic peptides derived from the propart region of human prorenin.

1. Five synthetic peptides which together spanned the propart segment of human prorenin were tested for their ability to interact with human renin, pepsin, gastricsin, cathepsin D, cathepsin E, calf chymosin and the aspartic proteinase from Endothia parasitica. 2. While two peptides showed no significant effect with any of the enzymes, a further two were cleaved by several enzymes. 3. Only one (corresponding to the 32P-43P residues in the propart sequence) acted as a weak competitive inhibitor of most of the enzymes.

Amino Acid Sequence↗
PubMed1992

Substrate specificity and inhibitors of aspartic proteinases.

Aspartic proteinases of vertebrate, fungal and retroviral origin have been characterised by utilisation of chromogenic peptide substrates and synthetic inhibitors as probes. By this means, the molecular topography of sub-sites within the active site cleft of individual enzymes has been elucidated. With suitable selection of residues to occupy each sub-site, the design of effective inhibitors specifically targetted against individual aspartic proteinases has become feasible.

Amino Acid Sequence↗
PubMed1991

Development of synthetic peptide substrates for the poliovirus 3C proteinase.

Picornaviruses, such as polio, translate their entire genome as a single polyprotein which must be proteolytically processed to produce the mature viral proteins. A majority of these cleavages are catalyzed by the virus-encoded cysteine proteinase, 3C. We report here the design and synthesis of a series of oligopeptide substrates, based upon native 3C cleavage sites, for an HPLC assay of poliovirus 3C proteinase activity. A similar series of peptides based upon human rhinovirus 3C cleavage sites was also examined. The enzyme shows a marked preference for those peptides with a proline in the P'2 position. A quenched fluorescent substrate suitable for continuous assay of 3C proteinase activity was also synthesized. Both the HPLC assay and the fluorescence assay were used to evaluate a number of potential 3C proteinase inhibitors.

3C Viral Proteases↗
PubMed1990

Sub-site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV-1.

A series of synthetic, chromogenic substrates for HIV-1 proteinase with the general structure Ala-Thr-His-Xaa-Yaa-Zaa*Nph-Val-Arg-Lys-Ala was synthesised with a variety of residues introduced into the Xaa, Yaa and Zaa positions. Kinetics parameters for hydrolysis of each peptide by HIV-1 proteinase at pH 4.7, 37 degrees C and u = 1.0 M were measured spectrophotometrically and/or by reverse phase FPLC. A variety of residues was found to be acceptable in the P3 position whilst hydrophobic/aromatic residues were preferable in P1. The nature of the residue occupying the P2 position had a strong influence on kcat (with little effect on Km); beta-branched residues Val or Ile in this position resulted in considerably faster peptide hydrolysis than when e.g. the Leu-containing analogue was present in P2.

Amino Acid Sequence↗
PubMed1990

Sensitive, soluble chromogenic substrates for HIV-1 proteinase.

By replacement of the P1' residue in a capsid/nucleocapsid cleavage site mimic with 4-NO2-phenylalanine (Nph), an excellent chromogenic substrate, Lys-Ala-Arg-Val-Leu*Nph-Glu-Ala-Met, for HIV-1 proteinase (kappa cat = 20 s-1, Km = 22 microM) has been prepared. Substitution of the Leu residue in P1 with norleucine, Met, Phe, or Tyr had minimal effects on the kinetic parameters (kappa cat and kappa cat/Km) determined at different pH values, whereas peptides containing Ile or Val in P1 were hydrolyzed extremely slowly. The spectrophotometric assay has been used to characterize the proteinase further with respect to pH dependence, ionic strength dependence, and the effect of competitive inhibitors of various types.

Amino Acid Sequence↗
PubMed1990

The selectivity of statine-based inhibitors against various human aspartic proteinases.

The interactions of five human enzymes (renin, pepsin, gastricsin, cathepsin D and cathepsin E) and the aspartic proteinase from Endothia parasitica with several series of synthetic inhibitors were examined. All of the inhibitors contained the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues in the P1-P1' positions. The residues occupying the peripheral sub-sites (P4 to P3') were varied systematically and inhibitory constants were determined for the interactions with each of the proteinases. Inhibitors were elucidated that specifically inhibited human renin and did not affect any of the other human enzymes or the fungal proteinase. With suitable selection of residues to occupy individual sub-sites, effective inhibitors of specific human aspartic proteinases may now be designed.

Amino Acid Sequence↗
PubMed1990

Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus.

The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1 micrograms/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).

Amino Acid Sequence↗