Biomedical subjects

S P Salowe

Publications and source records attributed to S P Salowe.

11 recordsLinked to original sources

PubMed2005

Anthrax lethal factor inhibition.

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) approximately 50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit "point of no return" model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

Animals↗

PubMed1998

Competitive and slow-binding inhibition of calcineurin by drug x immunophilin complexes.

The calcium- and calmodulin-activated protein phosphatase calcineurin (CN) is the target for the immunosuppressive drugs FK506 and cyclosporin A (CsA) when bound to their intracellular receptor proteins, the immunophilins known as FK506-binding protein (FKBP) and cyclophilin A (CypA), respectively. Investigation of the reaction kinetics for inhibition of CN using progress curves of [33P]phosphopeptide hydrolysis revealed slow-binding inhibition by the FK506 . FKBP complex. Final steady-state velocities were extracted by curve fitting over a range of substrate and inhibitor concentrations; the data fit well to a simple competitive inhibition model with a Ki of 14 nM for the FK506 . FKBP complex. The FKBP complex with L-732,531, an analog of FK506 containing a hydroxyethylindole substituent, was significantly more potent than FK506 x FKBP and was investigated in greater detail. The hyperbolic dependencies of the initial velocities and the first-order rate constants for the approach to steady state upon the concentration of L-732,531 x FKBP were consistent with a two-step inhibition mechanism in which the initial E x I complex slowly isomerizes to a more stable E x I* form. The reverse isomerization rate constant with L-732,531 . FKBP was markedly slower than that with FK506 x FKBP and is likely responsible for the higher affinity of the former for CN. Inhibition of CN by the CsA x CypA complex was not time-dependent, but the data did conform to a competitive inhibition model like FK506 x FKBP. These results are consistent with the hypothesis that both classes of drug x immunophilin complexes interact with a common locus on CN which excludes phosphopeptide binding in the enzyme's active site.

Amino Acid Sequence↗

PubMed1996

The utility of FK506-binding protein as a fusion partner in scintillation proximity assays: application to SH2 domains.

Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector in Escherichia coli as fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [3H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor-ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.

Amino Acid Sequence↗

PubMed1994

The NMR structure of the inhibited catalytic domain of human stromelysin-1.

The three-dimensional structure of the catalytic domain of stromelysin-1 complexed with an N-carboxyl alkyl inhibitor has been determined by NMR methods. The global fold consists of three helices, a five stranded beta-sheet and a methionine located in a turn near the catalytic histidines, classifying stromelysin-1 as a metzincin. Stromelysin-1 is unique in having two independent zinc binding sites: a catalytic site and a structural site. The inhibitor binds in an extended conformation. The S1' subsite is a deep hydrophobic pocket, whereas S2' appears shallow and S3' open.

Amino Acid Sequence↗

PubMed1993

Secondary structure and zinc ligation of human recombinant short-form stromelysin by multidimensional heteronuclear NMR.

Stromelysin-1, a member of the matrix metalloendoprotease family, is a zinc protease involved in the degradation of connective tissue in the extracellular matrix. As a step toward determining the structure of this protein, multidimensional heteronuclear NMR experiments have been applied to an inhibited truncated form of human stromelysin-1. Extensive 1H, 13C, and 15N sequential assignments have been obtained with a combination of three- and four-dimensional experiments. On the basis of sequential and short-range NOEs and 13C alpha chemical shifts, two helices have been delineated, spanning residues Asp-111 to Val-127 and Leu-195 to Ser-206. A third helix spanning residues Asp-238 to Gly-247 is characterized by sequential NOEs and 13C alpha chemical shifts, but not short-range NOEs. The lack of the latter NOEs suggests that this helix is either distorted or mobile. Similarly, sequential and interstrand NOEs and 13C alpha chemical shifts characterize a four-stranded beta-sheet with three parallel strands (Arg-100 to Ile-101, Ile-142 to Ala-147, Asp-177 to Asp-181) and one antiparallel strand (Ala-165 to Tyr-168). Two zinc sites have been identified in stromelysin [Salowe et al. (1992) Biochemistry 31, 4535-4540]. The NMR spectral properties, including chemical shift, pH dependence, and proton coupling of the imidazole nitrogens of six histidine residues (151, 166, 179, 201, 205, and 211), invariant in the matrix metalloendoprotease family, suggest that these residues are zinc ligands. NOE data indicate that these histidines form two clusters: one ligates the catalytic zinc (His-201, -205, and -211), and the other ligates a structural zinc (His-151, -166, and -179). Heteronuclear multiple quantum correlated spectra and specific labeling experiments indicate His-151, -179, -201, -205, and -211 are in the N delta 1H tautomer and His-166 is in the N epsilon 2H tautomer.

Amino Acid Sequence↗

PubMed1992

Characterization of zinc-binding sites in human stromelysin-1: stoichiometry of the catalytic domain and identification of a cysteine ligand in the proenzyme.

A determination of the zinc stoichiometry of the catalytic domain of the human matrix metalloproteinase stromelysin-1 has been carried out using enzyme purified from recombinant Escherichia coli that express C-terminally truncated protein. Atomic absorption spectrometry revealed that both the proenzyme (prostrom255) and the mature active form (strom255) contained nearly 2 mol of Zn/mol of protein. Full-length prostromelysin purified from a mammalian cell culture line also contained zinc in excess of 1 equiv. While zinc in prostrom255 could not be removed by dialysis against o-phenanthroline, similar treatment of mature strom255 resulted in the loss of one-half of the original zinc content. The peptidase activity of the zinc-depleted protein was reduced by greater than 85% but could be restored upon addition of Zn2+ or Co2+. Addition of a thiol-containing inhibitor to a CoZn hybrid enzyme resulted in marked spectral changes in both the visible and ultraviolet regions characteristic of sulfur ligation to Co2+. This direct evidence for an integral role in catalysis and inhibitor binding confirms the location of the exchangeable metal at the active site. To examine the environment of zinc in the proenzyme, a fully cobalt-substituted proenzyme was prepared by in vivo metal replacement. The absorbance features of dicobalt prostrom255 were consistent with metal coordination by the single cysteine present in the propeptide, although the data do not allow assignment to a particular zinc site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Elucidation of the order of oxidations and identification of an intermediate in the multistep clavaminate synthase reaction.

The enzyme clavaminate synthase (CS) catalyzes the formation of the first bicyclic intermediate in the biosynthetic pathway to the potent beta-lactamase inhibitor clavulanic acid. Our previous work has led to the proposal that the cyclization/desaturation of the substrate proclavaminate proceeds in two oxidative steps, each coupled to a decarboxylation of alpha-ketoglutarate and a reduction of dioxygen to water [Salowe, S. P., Marsh, E. N., & Townsend, C. A. (1990) Biochemistry 29, 6499-6508]. We have now employed kinetic isotope effect studies to determine the order of oxidations for CS purified from Streptomyces clavuligerus. By using (4'RS)-[4'-3H,1-14C]-rac-proclavaminate, a primary T(V/K) = 8.3 +/- 0.2 was measured from [3H]water release data, while an alpha-secondary T(V/K) = 1.06 +/- 0.01 was determined from the changing 3H/14C ratio of the product clavaminate. Values for the primary and alpha-secondary effects of 11.9 +/- 1.7 and 1.12 +/- 0.07, respectively, were obtained from the changing 3H/14C ratio of the residual proclavaminate by using new equations derived for a racemic substrate bearing isotopic label at both primary and alpha-secondary positions. Since only the first step of consecutive irreversible reactions will exhibit a V/K isotope effect, we conclude that C-4' is the initial site of oxidation in proclavaminate. As expected, no significant changes in the 3H/14C ratio of residual substrate were observed with [3-3H,1-14C]-rac-proclavaminate. However, two new tritiated compounds were produced in this incubation, apparently the result of isotope-induced branching brought about by the presence of tritium at the site of the second oxidation. One of these compounds was identified by comparison to authentic material as dihydroclavaminate, a stable intermediate that normally remains enzyme-bound. On the basis of the body of information available and the similarities to alpha-ketoglutarate-dependent dioxygenases, a comprehensive mechanistic scheme for CS is proposed to account for this unusual enzymatic transformation.

Anti-Bacterial Agents↗

PubMed1990

Purification and characterization of clavaminate synthase from Streptomyces clavuligerus: an unusual oxidative enzyme in natural product biosynthesis.

A pivotal step in the biosynthetic pathway to the beta-lactamase inhibitor clavulanic acid is the conversion of proclavaminic acid to clavaminic acid in a reaction requiring Fe2+, alpha-ketoglutarate, and oxygen [Elson, S. W., Baggaley, K. H., Gillett, J., Holland, S., Nicholson, N. H., Sime, J. T., & Woroniecki, S. R. (1987) J. Chem. Soc., Chem. Commun., 1736-1738]. Clavaminate synthase, the enzyme that catalyzes this oxidative cyclization/desaturation, has been purified to homogeneity from clavulanic acid producing cells of Streptomyces clavuligerus (ATCC 27064). The enzyme behaved as a monomer during gel filtration and migrated with Mr 47,000 during denaturing gel electrophoresis. After ion-exchange FPLC two active forms of the protein were resolved that differed slightly in kinetic constants and apparent molecular weight. Kinetic comparisons with the four possible diastereomers of proclavaminate confirmed the absolute configuration of the substrate to be 2S,3R. The stoichiometry of the overall transformation was determined to be proclavaminate + 2(alpha-ketoglutarate) + 2O2----clavaminate + 2(succinate) + 2CO2 + 2H2O. In the absence of proclavaminate a slow decarboxylation of alpha-ketoglutarate to succinate and CO2 was observed in an uncoupled reaction which resulted in enzyme inactivation. Steady-state kinetic studies were undertaken for an initial description of the enzyme's catalytic cycle. The double-reciprocal plot with alpha-ketoglutarate as the variable substrate was linear; this supports the proposal that two stepwise oxidations of proclavaminate occur, each with the consumption of alpha-ketoglutarate and oxygen and the release of succinate, CO2, and H2O. The intersecting initial velocity plots obtained from pairwise variation of substrate concentrations were consistent with a sequential kinetic mechanism for the first oxidation. Similarities observed between clavaminate synthase and alpha-ketoglutarate-dependent dioxygenases argue for a common mechanism of oxygen activation. However, the nature of the interactions of the substrates in the active site of clavaminate synthase apparently redirects the conventional hydroxylase activity of dioxygenases to the construction of a strained bicyclic skeleton driven by the overall reduction of dioxygen.

Aza Compounds↗

PubMed1987

Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate.

Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-deoxyuridine 5'-diphosphate (N3UDP). Incubation of RDPR with [3'-3H]N3UDP resulted in 0.2 mol of 3H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction. Incubation of RDPR with [beta-32P]N3UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N3UDP, but no azide release was detected. Analysis of the reaction of RDPR with [15N3]N3UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated. The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N3UDP as reported previously [Sjöberg, B.-M., Gräslund, A., & Eckstein, F. (1983) J. Biol. Chem. 258, 8060-8067], while the specific activity of the B1 subunit was reduced by half. Incubation of [5'-3H]N3UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme. Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.

Affinity Labels↗

PubMed1986

Cloning, overproduction, and purification of the B2 subunit of ribonucleoside-diphosphate reductase.

The nrdB gene, which encodes the B2 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1), was cloned into multicopy plasmid pSPS2. This vector, which contains the pL promoter of bacteriophage lambda and the tetracycline resistance gene of pBR322, was transformed into a lysogenic host with a thermolabile repressor. In the newly constructed strain, subunit B2 constituted approximately 25% of the soluble protein after heat induction, an overproduction of several hundredfold relative to the wild-type strain. Purification to homogeneity of the overproduced protein was accomplished by using DEAE and quaternary aminoethyl ion-exchange resins.

Amino Acid Sequence↗

PubMed1984

Porphobilinogen synthase modification with methylmethanethiosulfonate. A protocol for the investigation of metalloproteins.

A stable, reversibly sulfhydryl-modified, Zn2+-free porphobilinogen synthase (mod-apo-PBG synthase) has been prepared using methylmethanethiosulfonate. Mod-apo-PBG synthase prepared from holo-PBG synthase using [methyl-14C]methanethiosulfonate incorporated three thiomethyl groups/subunit. When apo-PBG synthase was prepared using EDTA alone, subsequent reaction with [methyl-14C]methanethiosulfonate resulted in incorporation of only two thiomethyl groups/subunit. Mod-apo-PBG synthase was catalytically inactive and contained less than 0.1 mol of Zn/mol of octameric enzyme; it could be reconstituted to full activity using 2-mercaptoethanol and Zn2+. A variety of metal ions were screened for their ability to reconstitute and/or reactivate mod-apo-PBG synthase. Only Zn2+ and Cd2+ reconstitute mod-apo-PBG synthase to full activity. When comparing mod-apo-PBG synthase prepared from holo-PBG synthase in the presence of EDTA with mod-apo-PBG synthase prepared from holo-PBG synthase in the absence of EDTA, no difference was detected in either Zn content, stoichiometry of 14C-labeling, or kinetic behavior. We have confirmed both the observations that four Zn2+/mol of octameric apoenzyme are necessary for full catalytic activity and that holoenzyme, isolated in the presence of 10 microM ZnCl2, contains eight Zn2+/octamer. The additional four binding sites are not catalytically important. Methylmethanethiosulfonate modification is presented as a generally useful method for the investigation of metalloproteins because it provides a route for the preparation of stable apoproteins and a direct method for metal ion replacement.

Animals↗