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Featured researches published by Dieter Brömme.


Journal of Enzyme Inhibition | 1989

Peptide Methyl Ketones as Reversible Inhibitors of Cysteine Proteinases

Dieter Brömme; Beate Bartels; Heidrun Kirschke; Siegfried Fittkau

Peptide methyl ketones represent a new class of reversible, competitive cysteine proteinase inhibitor with little or no effect on serine proteinases. The affinity of the inhibitors to papain (EC 3.4.22.3), cathepsin B (EC 3.4.22.1) and cathepsin L (EC 3.4.22.15) depends on the peptide chain length and on side-chain effects. Variations in the P1 and P4 positions (terminology of Schechter and Berger) and their influence on the efficiency of the inhibitors have been investigated. The most effective inhibitors display inhibition constants in the micromolar range. In contrast to the endopeptidases papain and the cathepsins B and L, the aminoendopeptidase cathepsin H (EC 3.4.22.16) is not inhibited by N-acylated peptide methyl ketones but only by amino methyl ketones containing a free alpha-amino group. The endopeptidases are not affected by amino methyl ketones.


Biochimica et Biophysica Acta | 1996

N-peptidyl, O-acyl hydroxamates: Comparison of the selective inhibition of serine and cysteine proteinases

Hans-Ulrich Demuth; Angelika Schierhorn; Philip Bryan; Ralph Höfke; Heidrun Kirschke; Dieter Brömme

Two series of N-aminoacyl, O-benzoyl hydroxamates were designed to investigate the influence of the substituted benzoyl residue on the hydrolytic stability and the reactivity of these potential inhibitors towards selected cysteine and serine proteinases. The inactivators react more rapidly with cysteine proteinases than with the serine enzymes tested. While Z-Phe-Gly-NHO-Nbz is the most reactive inhibitor of cathepsin L, inhibiting the target protein by a second order rate constant of 932.000 M-1 s-1, the bacterial serine proteinase thermitase is inhibited best by Z-Gly-Phe-NHO-Nbz, exhibiting a second-order rate constant of 1.170 M-1 s-1. Thiolsubtilisin, having the thiol-group as the reactive nucleophile instead of serine, exhibits specificity constants of the inactivation two orders of magnitude smaller than subtilisin. The degree of selectivity of the inhibitors relative to cathepsin B, cathepsin L, cathepsin S and papain varies up to two orders of magnitude with respect to their second order rate constant of inactivation. The inhibitory reactivity of these compounds varies only up to sixfold depending on the benzoyl substituent. Similarly, the rate constants for the hydrolytic decomposition of the compounds vary by a factor of about 6, suggesting that the structural and mechanistic features of the compounds which are responsible for decomposition as well as for the enzyme inhibition are the same. Comparing both reactions, the data allow the calculation of an acceleration factor of 2.4 x 10(10) for the inhibition of cathepsin L by its most effective inhibitor, clearly characterizing this enzyme inhibition reaction as enzyme-activated.


FEBS Letters | 1993

N‐Peptidyl‐O‐carbamoyl amino acid hydroxamates: Irreversible inhibitors for the study of the S2′ specificity of cysteine proteinases

Dieter Brömme; Heidrun Kirschke

A series of new inhibitors for cysteine proteinases with the general structure Z‐Phe‐Gly‐NHO‐CO‐Aa (Aa = amino acids) was synthesized and tested as inhibitors of papain‐like enzymes (cathepsins S, L, B and papain). Like N‐peptidyl‐O‐acyl hydroxamates the inhibitors inactivate cysteine proteinases by a sulfenamidation of the active site cysteine residue. The most effective inhibitors display second order‐rate constants of inactivation in the range of 103–104 M−1·s−1. Since the structure of the N‐peptidyl‐O‐carbamoyl amino acid hydroxamates allows the variation of the leaving group this class of inhibitors was used as a new tool for the evaluation of the S2′ specificity of cysteine proteinases.


Methods in Enzymology | 1994

[48] N,O-Diacyl hydroxamates as selective and irreversible inhibitors of cysteine proteinases

Dieter Brömme; Hans-Ulrich Demuth

Publisher Summary This chapter examines N, O -diacyl hydroxamates as selective and irreversible inhibitors of cysteine proteinases. Cysteine proteases represent attractive targets for the design of inhibitors as the proteinases play a role in degenerative diseases and in malignant tumor and parasite development. Inhibitors of cysteine proteases consist of a peptide part binding to the S specificity subsites of the target proteinase and a reactive group interacting with a catalytic active site residue. N, O -diacyl hydroxamates is a novel class of peptide-derived inhibitors, which permits variations of the N -acyl and O -acyl residues and, thus, a selective control of their affinity and reactivity toward the enzymes. N, O -diacyl hydroxamates are easily accessible synthetically. N, O -diacyl hydroxamates are stable under acidic conditions however less stable toward strong alkali. N, O -Diacyl hydroxamates react irreversibly with their target cysteine proteinase. No recovery of activity of the enzyme is achieved after the removal of the excess inhibitor. The irreversibility of inactivation can be confirmed by gel-filtration or dilution experiments.


Current Microbiology | 1984

Substrate specificity of thermitase, a thermostable serine proteinase fromThermoactinomyces vulgaris

Dieter Brömme; Rolf Kleine

The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu5-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln4-His5, Ser9-His10, Leu11-Val12, Leu15-Tyr16 and in the oxidized insulin A chain at Cys SO3H11-Ser12, Leu13-Tyr14, and Leu16-Glu17 were observed. Additional cleavages of the bonds His5-Leu6, Arg22-Gly23, Phe24-Phe25, Phe25-Tyr26, and Tyr26-Thr27 in the oxidized B chain and Cys SO3H6-Cys SO3H7 and Tyr19-Cys SO3H20 in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg5-Ser6, Ser6-Ser7, Arg18-Val19, and Gly27-Gly28. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins.


Biochimica et Biophysica Acta | 1993

Novel N-peptidyl-O-acyl hydroxamates : selective inhibitors of cysteine proteinases

Dieter Brömme; Ulf Neumann; Heidrun Kirschke; Hans-Ulrich Demuth

A series of N-peptidyl-O-acyl hydroxamates with a lysine in P1 was synthesized and tested as inactivators of lysosomal cysteine proteinases (cathepsins S, L, B and H) and trypsin-like serine proteinases (trypsin, thrombin, plasmin, t-PA). N-peptidyl-O-acyl hydroxamates were shown to be selective inhibitors of cysteine proteinases. With the exception of cathepsin H, the lysosomal cysteine proteinases were inactivated 2-5 orders of magnitude more rapidly than serine proteinases with a comparable primary substrate specificity. The highest second-order rate constants of inactivation for the cysteine proteinases are in the range of 10(5)-10(6) M-1 s-1. The order of inhibitor specificity for the cysteine proteinases is comparable to the enzymes substrate specificity.


Current Microbiology | 1984

Use of microbial peptide inhibitors for characterization of the substrate specificity of thermitase, a thermostable serine protease fromThermoactinomyces vulgaris

Dieter Brömme; Rolf Kleine

Seven microbial peptide inhibitors—chymostatin, antipain, elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon—were tested for their efficiency to inhibit thermitase, a thermostable serine protease fromThermoactinomyces vulgaris. Chymostatin and antipain were the most effective inhibitors, with Ki values of 7×10−8M and 2×10−7M, respectively. Except for leupeptin, all inhibitors resist hydrolysis by thermitase. Leupeptin, however, is cleaved by thermitase between the two leucylresidues. Further, a close relationship in specificity between thermitase and subtilisin BPN′ and their distinct discrimination from elastase specificity was demonstrated by using these inhibitors.


FEBS Letters | 1987

Action of rat liver cathepsin B on bradykinin and on the oxidized insulin A-chain

Dieter Brömme; Albert Steinert; Siegfried Fittkau; Heidrun Kirschke

Rat liver cathepsin B was tested for its peptide‐bond specificity against bradykinin and the oxidized insulin A‐chain. Bradykinin was shown to be resistant to the action of cathepsin B. One possible reason for this resistance is the proline content of the peptide and the discrimination against proline residues at three or four subsites of cathepsin B. Oxidized insulin A‐chain was degraded by a peptidyl dipeptidase activity. Three dipeptides were cleaved from the C‐terminal part of the insulin A‐chain after having been incubated for 2 h (molar ration E:S = 1:2800) and six dipeptides were released after a longer digestion (10 h, E:S = 1:575).


Biochemical Journal | 1989

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins

Heidrun Kirschke; Bernd Wiederanders; Dieter Brömme; Ari Rinne


Biochemical Journal | 1989

The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B.

Dieter Brömme; A Steinert; S Friebe; Siegfried Fittkau; B Wiederanders; Heidrun Kirschke

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Philip Bryan

Biotechnology Institute

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Ari Rinne

University of Tromsø

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