Biomedical subjects

C A Kettner

Publications and source records attributed to C A Kettner.

34 records · Page 2Linked to original sources

PubMed1992

Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

In vivo characterization of a new synthetic thrombin inhibitor.

We have examined the in vivo pharmacology of DuP 714 (Ac-[D]-Phe-Pro-boroArginine), a representative of a new series of synthetic thrombin inhibitors which contain a boronic acid derivative of arginine. Intravenous bolus injections of DuP 714 in anesthetized rats and conscious rabbits produced transient elevations of clotting times. Clinically relevant prolongations of the APTT were also observed in rabbits after i.v. infusion of less than 0.1 mg kg-1 h-1. Efficacy against venous thrombosis was demonstrated in a rabbit model of stasis induced thrombosis. Clots formed in 100% of control animals and only 33% of animals treated with 0.5 mg/kg DuP 714, and were less severe in treated animals. In a rabbit arterial-venous shunt model mimicking arterial thrombosis, occlusion occurred within 30 min in 72% of control animals vs. 11% of animals treated with 0.1 mg kg-1 h-1 DuP 714. Results indicate that DuP 714 is a highly effective anticoagulant which should be useful for the prevention of both venous and arterial thrombotic diseases.

Animals↗

PubMed1992

A phosphinic acid dipeptide analogue to stabilize peptide drugs during their intranasal absorption.

A major challenge in intranasal delivery of peptides is to overcome the enzymatic barrier that limits their absorption. Aminopeptidase inhibitors may be useful for improving systemic delivery of peptide drugs administered nasally. A phosphinic acid dipeptide analogue, a transition-state analogue aminopeptidase inhibitor in which the phosphinate moiety exists in a tetrahedral state mimicking peptides during their enzymatic hydrolysis, was synthesized and tested nasally in situ in rats. This inhibitor was found to inhibit greatly the degradation of the model peptide leucine-enkephalin in the nasal perfusate at less than or equal to 2 microM concentrations. The nasal peptidase hydrolytic activity was reversible after exposure to the inhibitor. This inhibitor has the advantage of efficacy at very low concentrations and reversibility of effects.

Absorption↗

PubMed1992

Human immunodeficiency virus type 1 and type 2 protease monomers are functionally interchangeable in the dimeric enzymes.

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 proteases are dimers of identical subunits. We made a construct for the expression of recombinant one-chain HIV-2 protease dimer, which, like the previously described one-chain HIV-1 protease dimer, is fully active. The constructs for the one-chain dimers of HIV-1 and HIV-2 proteases were modified to produce hybrid one-chain dimers consisting of both HIV-1 and HIV-2 protease monomers. Although the monomers share only 47.5% sequence identity, the hybrid one-chain dimers are fully active, suggesting that the folding of both HIV-1 and HIV-2 protease monomers is functionally similar.

Base Sequence↗

PubMed1991

Structural basis for broad specificity in alpha-lytic protease mutants.

Binding pocket mutants of alpha-lytic protease (Met 192----Ala and Met 213----Ala) have been constructed recently in an effort to create a protease specific for Met just prior to the scissile bond. Instead, mutation resulted in proteases with extraordinarily broad specificity profiles and high activity [Bone, R., Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-195]. To understand the structural basis for the unexpected specificity profiles of these mutants, high-resolution X-ray crystal structures have been determined for complexes of each mutant with a series of systematically varying peptidylboronic acids. These inhibitory analogues of high-energy reaction intermediates provide models for how substrates with different side chains interact with the enzyme during the transition state. Fifteen structures have been analyzed qualitatively and quantitatively with respect to enzyme-inhibitor hydrogen-bond lengths, buried hydrophobic surface area, unfilled cavity volume, and the magnitude of inhibitor accommodating conformational adjustments (particularly in the region of another binding pocket residue, Val 217A). Comparison of these four parameters with the Ki of each inhibitor and the kcat and Km of the analogous substrates indicates that while no single structural parameter consistently correlates with activity or inhibition, the observed data can be understood as a combination of effects. Furthermore, the relative contribution of each term differs for the three enzymes, reflecting the altered conformational energetics of each mutant. From the extensive structural analysis, it is clear that enzyme flexibility, especially in the region of Val 217A, is primarily responsible for the exceptionally broad specificity observed in either mutant. Taken together, the observed patterns of substrate specificity can be understood to arise directly from interactions between the substrate and the residues lining the specificity pocket and indirectly from interactions between peripheral regions of the protein and the active-site region that serve to modulate active-site flexibility.

Alanine↗

PubMed1991

Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function.

Dipeptidyl peptidase IV (DP-IV; dipeptidyl-peptide hydrolase, EC 3.4.14.5) is a serine protease with a specificity for cleaving Xaa-Pro dipeptides from polypeptides and proteins. It is found in a variety of mammalian cells and tissues, including those of lymphoid origin where it is found specifically on the surface of CD4+ T cells. Although the functional significance of this enzyme has not been established, a role in T-cell activation and immune regulation has been proposed. Here we report that Ala-boroPro and Pro-boroPro, where boroPro is the alpha-amino boronic acid analog of proline, are potent and specific inhibitors of DP-IV, having Ki values in the nanomolar range. Blocking the N terminus of Ala-boroPro abolishes the affinity of this inhibitor for DP-IV, while removal of the N-terminal residue, to give boroPro, reduces the affinity for DP-IV by 5 orders of magnitude. The dipeptide boronic acids exhibit slow-binding kinetics, while boroPro does not. We also report here that low concentrations of Pro-boroPro inhibit antigen-induced proliferation and interleukin 2 production in murine T-cell lines but do not inhibit the response of these T cells to the mitogen concanavalin A. These results indicate that DP-IV plays a role in antigen-induced, but not mitogen-induced, activation of T lymphocytes.

Animals↗

PubMed1990

High-level synthesis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies.

A complete chemical synthesis and assembly of genes for the production of human immunodeficiency virus type-I protease (HIV-PR) and its precursors are described. The T7 expression system was used to produce high levels of active HIV-PR and its precursors in Escherichia coli inclusion bodies. The gene encoding the open reading frames of HIV-PR was expressed in E. coli as a 10-kDa protein, while the genes encoding HIV-PR precursors were expressed as larger proteins, which were partially processed in E. coli to the 10-kDa form. These processing events are autoproteolytic, since a single-base mutation, changing the active-site aspartic acid to glycine, completely abolished the conversion. HIV-PR can be released with 8 M urea from washed cellular inclusion bodies, resulting in a preparation with few bacterial host proteins. After refolding, this preparation contains no nonspecific protease or peptidase activities. The recombinant HIV-PR isolated from inclusion bodies cleaves HIV-PR substrates specifically with a specific activity comparable to column-purified HIV-PR.

Amino Acid Sequence↗

PubMed1990

Inhibition of IgA1 proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids.

The alpha-aminoboronic acid analog of proline has been synthesized and incorporated into a number of peptides as the COOH-terminal residue. These peptide prolyl boronic acids are potent inhibitors of both the type 1 and type 2 IgA proteinases from Neisseria gonorrhoeae and Hemophilus influenzae, but not of the functionally similar IgA proteinase from Streptococcus sanguis. The best inhibitors synthesized thus far have Ki values in the nanomolar range (4.0 to 60 nM). These results indicate that the N. gonorrhoeae and the H. influenzae enzymes belong to the serine protease family of proteolytic enzymes while that from S. sanguis does not. As a group, the IgA proteinases have been noted for their remarkable specificity; thus, the peptide prolyl boronic acids reported here are the first small synthetic molecules to exhibit a relatively high affinity for the active site of an IgA proteinase and are therefore the first to yield some insight into the active site structure and specificity requirements of these enzymes.

Amino Acid Sequence↗

PubMed1990

Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer.

A single-chain tethered dimer of human immunodeficiency virus protease (HIV-PR) was produced by expression of a synthetic gene in Escherichia coli. The tethered dimer, which consists of two 99-amino acid HIV-PR subunits linked together by a pentapeptide, was isolated from inclusion bodies and refolded as an active protease with enzymatic properties very similar to those of the natural dimer at pH 5.5. In addition to demonstrating that the tethered dimer is active, we have shown that the tethered dimer is more stable than the natural HIV-PR dimer at pH 7.0. This is attributed to dissociation of the natural HIV-PR dimer, for which a surprisingly high dissociation constant, 5 X 10(-8) M was measured. Furthermore, the tethered dimer offers an opportunity to produce asymmetric dimer mutants and thereby determine the effect of changes in one of the two subunits on protease activity. In one such mutant, a single active-site aspartic residue was changed to a glycine residue. This protein was inactive, consistent with a requirement for an aspartic residue from each subunit to constitute an active site of HIV-PR.

Amino Acid Sequence↗

PubMed1989

Structural analysis of specificity: alpha-lytic protease complexes with analogues of reaction intermediates.

To better understand the structural basis of enzyme specificity, the structures of complexes formed between alpha-lytic protease, an extracellular serine protease of Lysobacter enzymogenes, and five inhibitory peptide boronic acids (R2-boroX, where R2 is methoxysuccinyl-Ala-Ala-Pro- and boroX is the alpha-aminoboronic acid analogue of Ala, Val, Ile, Norleu, or Phe) have been studied at high resolution by X-ray crystallography. The enzyme has primary specificity for Ala in the P1 position of peptide substrates with catalytic efficiency decreasing with increasing side-chain volume. Enzyme affinity for inhibitors with boroVal, boroIle, and boroPhe residues is much higher than expected on the basis of the catalytic efficiencies of homologous substrates. Covalent tetrahedral adducts are formed between the active-site serine and the boronic acid moieties of R2-boroAla, R2-boroVal R2-boroIle, and R2-boroNorleu. Though R2-boroVal is a slowly bound inhibitor and R2-boroAla is rapidly bound [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry 27, 7682-7688], there appear to be no structural differences that could account for slow binding. The removal from solution of 20% more hydrophobic surface on binding accounts for the improved affinity of alpha-lytic protease for R2-boroVal relative to R2-boroAla. The high affinity of the enzyme for R2-boroIle derives from the selective binding of the L-allo stereoisomer of the boroIle residue, which can avoid bad steric interactions in the binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1989

Crystal versus solution structure of enzymes: NMR spectroscopy of a peptide boronic acid-serine protease complex in the crystalline state.

The effectiveness of boronic acids as inhibitors of serine proteases has been widely ascribed to the ability of the boronyl group to form a tetrahedral adduct with the active-site serine that closely mimics the putative tetrahedral intermediate or transition state formed with substrates. However, recent 15N NMR studies of alpha-lytic protease (EC 3.4.21.12) in solution have shown that some boronic acids and peptide boronic acids form adducts with the active-site histidine instead of with the serine. Such histidine-boron adducts have not thus far been reported in x-ray diffraction studies of boronic acid-serine protease complexes. Here, we report an 15N NMR study of the MeOSuc-Ala-Ala-Pro-boroPhe complex of alpha-lytic protease in the crystalline state using magic-angle spinning. Previous 15N NMR studies have shown this complex involves the formation of a histidine-boron bond in solution. The 15N NMR spectra of the crystalline complex are essentially identical to those of the complex in solution, thereby showing that the structure of this complex is the same in solution and in the crystal and that both involve formation of a histidine-boron adduct.

Amino Acid Sequence↗

PubMed1988

Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids.

The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1988

Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes.

15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.

Binding Sites↗

PubMed1988

Interaction of peptide boronic acids with elastase: circular dichroism studies.

Boronic acid derivatives of good peptide substrates of the serine proteases cause slow-binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol Chem. 259:15106-15114, 1984). These inhibitors are thought to act as reaction-intermediate analogs. Three peptide boronic acids--Ac-Pro-boro-Val-OH, DNS-Ala-Pro-boro-Val-OH, and Ac-Ala-Ala-Pro-boro-Val-OH--were chosen for far-ultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitor (Ki = 0.27 microM) and the latter are slow-binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel beta-sheet by the peptide inhibitor itself indicate that there is no significant change in the secondary structure of the enzyme in either the initial or final inhibitor complex. A kinetic experiment confirmed that the slow-binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the slow binding.

Animals↗

PubMed1987

Serine protease mechanism: structure of an inhibitory complex of alpha-lytic protease and a tightly bound peptide boronic acid.

The structure of the complex formed between alpha-lytic protease, a serine protease secreted by Lysobacter enzymogenes, and N-tert-butyloxycarbonylalanylprolylvaline boronic acid (Ki = 0.35 nM) has been studied by X-ray crystallography to a resolution of 2.0 A. The active-site serine forms a covalent, nearly tetrahedral adduct with the boronic acid moiety of the inhibitor. The complex is stabilized by seven hydrogen bonds between the enzyme and inhibitor with additional stabilization arising from van der Waals interactions between enzyme and inhibitor side chains and the burying of 330 A2 of hydrophobic surface area. Hydrogen bonding between Asp-102 and His-57 remains intact in the enzyme-inhibitor complex, and His N epsilon 2 is well positioned to donate its hydrogen to the leaving group. Little change in the positions of protease residues was observed on complex formation (root mean square main chain deviation = 0.13 A), suggesting that in its native state the enzyme is complementary to tetrahedral reaction intermediates or to the nearly tetrahedral transition state for the reaction.

Boronic Acids↗

PubMed1984

Inhibition of the serine proteases leukocyte elastase, pancreatic elastase, cathepsin G, and chymotrypsin by peptide boronic acids.

Three alpha-aminoboronic acid-containing analogs of good peptide substrates for serine proteases were synthesized, MeO-Suc-Ala-Ala-Pro-boro-Phe-OH, MeO-Suc-Ala-Ala-Pro-boro-Ala-OH, and MeO-Suc-Ala-Ala-Pro-boro-Val-OH. They were effective inhibitors of chymotrypsin, cathepsin G, and both leukocyte and pancreatic elastase at nanomolar concentrations (0.10-20 nM). Except for cathepsin G, inhibition was not simply competitive, but showed kinetic properties corresponding to the mechanism for slow-binding inhibition, i.e. E + I in equilibrium EI in equilibrium EI*, where EI and EI* are enzyme-inhibitor complexes and EI* is more stable than EI. This type of inhibition has not been observed previously for synthetic inhibitors or serine proteases and in this study it was observed only for peptide boronic acids which satisfy the primary specificity requirements of the protease.

Animals↗