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H U Demuth

Publications and source records attributed to H U Demuth.

At least 37 records · Page 2Linked to original sources
PubMed1994

Inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydroxyl- amine: formation of a covalent enzyme-inhibitor linkage in the form of a carbamate derivative.

The mechanism of inactivation of serine proteases by N-peptidyl-O-aroylhydroxylamines was studied by X-ray crystallography. Cocrystals of subtilisin Carlsberg inactivated with N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-nitrobenzoy lhydroxylamine were grown, and diffraction data to 1.8-A resolution were obtained. The resulting electron density maps clearly reveal that the gamma-oxygen of the catalytic serine forms a carbamate derivative with the inhibitor. The peptide part of the inhibitor does not form the usual antiparallel beta-sheet in the P binding cleft but protrudes out of the active site and is stabilized by a network of water molecules. These results, combined with kinetic characterization reported previously [Demuth, H.-U., Schoenlein, C., & Barth, A. (1989b) Biochim. Biophys. Acta 996, 19-22; Schmidt, C., Schmidt, R., & Demuth, H.-U. (1990) Peptides (Giralt, E., & Andreu, D., Eds.) ESCOM Science Publishers B.V., Amsterdam] support the existence of at least one intermediate between the formation of the Michaelis complex and the final product. We suggest a mechanism for the inactivation of subtilisin Carlsberg by N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydr oxylamine whereby a negatively charged Michaelis complex undergoes a Lossen rearrangement giving rise to an isocyanate intermediate that reacts with the side chain of the active site serine.

Amino Acid Sequence↗
PubMed1993

Inhibition of aminopeptidases by N-aminoacyl-O-4-nitrobenzoyl hydroxamates.

10 N-aminoacyl-O-4-nitrobenzoyl hydroxamates were investigated as potential inhibitors of aminopeptidases. While the metal-depending enzymes aminopeptidase M, aminopeptidase P and leucine aminopeptidase were inhibited reversibly by the compounds, the thiol enzyme cathepsin H was inhibited efficiently in time-dependent reactions according to its substrate specificity. N-phenylalanyl-O-4-nitrobenzoyl hydroxamate was shown to be most effective, exhibiting a second-order-rate constant of inhibition of 31,766 M-1 s-1.

Amino Acid Sequence↗
PubMed1993

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

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 enzyme's substrate specificity.

Amino Acid Sequence↗
PubMed1993

Design of (omega-N-(O-acyl)hydroxy amid) aminodicarboxylic acid pyrrolidides as potent inhibitors of proline-specific peptidases.

A novel class of competitive, acylating inhibitors for the proline-specific peptidases: dipeptidyl peptidase IV, dipeptidyl peptidase II and prolyl endopeptidase, has been developed. The inhibitor molecules combine the efficacy of aminoacyl pyrrolidides and the potential transacylating capability of diacyl hydroxyl amines. The N-terminal deblocked inhibitors are potent reversible inhibitors of porcine kidney dipeptidyl peptidase IV, human placenta dipeptidyl peptidase II exhibiting Ki values in the microM range. Boc-protected (omega-N-hydroxy acyl amid) aminodiacarboxylic acid pyrrolidides inhibit substrate hydrolysis by prolyl endopeptidases from different sources competitively reaching Ki values of 30 nM to 60 microM. Additionally, alpha-N-BOC-(omega-N-hydroxy acetyl) glutaminyl pyrrolidide modifies human placenta prolyl endopeptidase in a time-dependent reaction.

Amino Acid Sequence↗
PubMed1993

Enzymatic activity of CD26 (dipeptidylpeptidase IV) is not required for its signalling function in T cells.

CD26 is a proteolytic enzyme (dipeptidylpeptidase IV) expressed on the T cell surface that defines an alternative activation signal for human T lymphocytes. Crosslinking of CD26 via monoclonal antibodies triggers proliferation and cytotoxicity in preactivated T cells. In this study, we used highly specific competitive and irreversible inhibitors of dipeptidylpeptidase IV to study the role of the enzymatic activity in activation of CD26-transfected T cells as well as of CD26-expressing normal human T cell clones. These inhibitors at concentrations that blocked up to 95% of the enzymatic activity, did not specifically inhibit T cell activation neither via TCR/CD3 nor via CD26 itself. This demonstrates that the enzymatic activity of CD26 is not required for its T cell activating properties.

Animals↗
PubMed1993

Inhibition of dynorphin converting enzymes from human spinal cord by N-peptidyl-O-acyl hydroxylamines.

Two cysteine proteinases, cleaving dynorphins A and B to enkephalins, were isolated from the human spinal cord. These enzymes were found to be competitively inhibited by a new class of synthetic inhibitors: N-peptidyl-O-acyl hydroxylamines. The most potent (Ki < 20 microM) were the N-terminally protected peptides Z-Phe-Phe-NHO-Ma and Boc-Phe-Gly-NHO-Bz, both containing hydrophobic amino acids at the P2 position. N-Peptidyl-O-acyl hydroxylamines were converted in water solution to the corresponding hydroxamic acids and no cleavage of the peptide bond within the inhibitor sequence was observed after prolonged incubation with the enzymes. It is anticipated that these synthetic compounds may serve as potential pharmacological tools for in vitro studies on dynorphin processing.

Amino Acid Sequence↗
PubMed1993

Peptidyl ammonium methyl ketones as substrate analog inhibitors of proline-specific peptidases.

Prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DP IV) are serine enzymes cleaving highly specific prolyl peptide bonds. Both enzymes were found to be inhibited by newly designed peptidyl ammonium and pyridinium methyl ketones acting as slow binding inhibitors. The most potent inhibitor of PEP is Z-Pro-Pro-CH2N+C5H5 exhibiting a Ki* value of 1.8 nM with a first-order rate constant of Kon 0.0022 s-1 for the formation of the tight enzyme-inhibitor complex. DP IV and H-Pro-Pro-CH2N+ (CH3)3 form an enzyme-inhibitor-complex with an apparent second order rate constant of 2713 M-1 s-1. In contrast to the very stable N-terminal protected Z-Pro-Pro-CH2N+ (CH3)3, the deblocked derivative decomposes rapidly in aqueous solution.

Dipeptidyl Peptidase 4↗
PubMed1992

Relationship between primary structure and activity in exorphins and endogenous opioid peptides.

We have found a correlation between the certain characteristics of primary structure and biologic activity in exorphins and endogenous opioid peptide family. The characteristics of primary structure are the content of certain segment pairs as well as the density of their arrangement in a peptide. These segment pairs represent basic elements of the regulatory peptide primary structure pattern, which was found recently [Dokl. Akad. Nauk USSR 289 (1986) 721-724; Int. J. Peptide Prot. Res. 38 (1991) 505-510].

Amino Acid Sequence↗
PubMed1992

Characterization of dipeptidyl peptidase IV (CD26) from human lymphocytes.

The membrane-bound dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) has been purified 5,400-fold from human peripheral blood mononuclear cells. The purification procedure included detergent solubilization and successive chromatography on DEAE Sepharose Fast Flow, Con A Sepharose, Cu2+ loaded metal-chelating Sepharose, Sephacryl S-300 High Resolution and Q Sepharose Hiload. The molecular mass of the native, detergent solubilized enzyme estimated by gel filtration was 264.kDa. Chromatofocusing indicated a pI of approximately 5.0. The pI optimum was 8.7. The enzymatic activity of the purified preparation was irreversibly inhibited by N-(H-Phe-Pro)-O-(4-nitrobenzoyl)hydroxylamine hydrochloride in the micromolar range. The binding of purified DPP IV to CD26 monoclonal antibodies confirmed the identity between CD26 and dipeptidyl peptidase IV. The purification and characterization of lymphocytic dipeptidyl peptidase IV is of great value for the identification of its natural substrates and for the study of its physiological significance in the T-lymphocyte function.

Amino Acid Sequence↗
PubMed1991

Dipeptidyl peptidase IV in the immune system. Effects of specific enzyme inhibitors on activity of dipeptidyl peptidase IV and proliferation of human lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase playing a significant role in the process of activation and proliferation of human thymus-derived lymphocytes. This conclusion is drawn from (1) the induction of this enzyme on mitogen-activated T lymphocytes (cf. Schön, E. & Ansorge, S. (1990) Biol. Chem. Hoppe-Seyler 371, 699-705) and (2) the impairment of different functions of activated T cells in the presence of specific inhibitors and antibodies against DP IV (Schön, E. & al. (1987) Eur. J. Immunol 17, 1821-1826). This paper is aimed at testing new active site-specific peptide inhibitors for their efficiency as inhibitors of lymphocyte DP IV and DNA synthesis of mitogen-stimulated lymphocytes. These inhibitors comprise (i) diacylhydroxylamine derivatives of Xaa-Pro or Xaa-Ala peptides, (ii) different oligopeptides with N-terminal Xaa-Pro-sequences, and (iii) amino-acid amides of the pyrrolidide and the thiazolidide type. The thiazolidides of epsilon-(4-nitrobenzyloxycarbonyl)-L-lysine and of L-isoleucine as well as Ala-Pro-nitrobenzoylhydroxylamine are the most effective inhibitors in both test systems, yielding half-maximal inhibitory concentrations in the micromolar range. Cell viability was not impaired in this effective concentration range. Other inhibitors of DP IV are one to two orders of magnitude less efficient in the suppression of lymphocyte proliferation.

Binding Sites↗
PubMed1991

Inhibition of proteases with enkephalin-analogue inhibitors.

N-peptidyl-O-acyl hydroxylamines have proven to be effective and selective mechanism-based inhibitors of serine and cysteine proteases as demonstrated using enzymes with specificities for hydrophobic amino acids at the cleavage site. Here, we report for the first time the inhibition of proteases able to accommodate cationic amino acid side chains in their binding pockets using compounds of this inhibitor class. Trypsin and papain are inactivated by enkephalin-analogue diacyl hydroxylamines in a time-dependent and irreversible manner exhibiting second-order rate constants in the range of 100-1000 M-1.s-1. In contrast, human cerebrospinal fluid dynorphin-converting enzyme (hCSFDCE) is inhibited only moderately by these inhibitors. Mechanistic implications have been derived.

Amino Acid Sequence↗
PubMed1991

Studies of specificity and inhibition of human cerebrospinal fluid dynorphin converting enzyme.

Dynorphin-converting activity was recently discovered in human cerebrospinal fluid. This enzyme (hCSF-DCE) cleaves dynorphin A, dynorphin B and alpha-neoendorphin to release Leu-enkephalin-Arg6. To characterize the enzyme further we used several protease inhibitors, including N-peptidyl-O-acyl hydroxylamines which are known to act as potent irreversible inhibitors of serine and cysteine proteinases. No irreversible inactivation occurred but strong, reversible effects on the dynorphin-converting activity by some of the inhibitors tested could be observed. Although, hCSF-DCE binds its substrates (dynorphin A and B) in the microM-mM concentration range, it exhibits high specificity in recognizing and cleaving the linkage between the two basic amino acids in the substrate sequence.

Amino Acid Sequence↗
PubMed1991

Reaction of dipeptidylpeptidase IV with substrate-analogous azapeptides.

The reaction of dipeptidyl peptidase IV (EC 3.4.14.5.) with azapeptide substrates containing azaalanine or azaproline in the P1-position was investigated. Accumulation of a fairly stable acyl-enzyme could be shown for ester substrates. Ala-AzaPro-pNA is a very poor substrate of DP IV and does not accumulate an acyl-enzyme. DP IV does not react with active-site titrants for trypsin-like serine proteases.

Amino Acid Sequence↗
PubMed1990

Recent developments in inhibiting cysteine and serine proteases.

Some 20 years ago, affinity labelling was introduced to help gather information on active-site catalytic groups and the mechanisms of proteolytic enzymes. Now this knowledge is used to produce specific and selective inhibitors for these enzymes. The concept of "biospecific drug design" has stimulated progress in turning the inhibitors into therapeutically applicable agents. For instance, sales of antihypertensive drugs based on inhibitors of the angiotensin converting enzyme are expected to be over 2 billion US-$ in 1992. This partly illustrates the efforts made by many researchers to introduce strategies in potease inhibition for medicinal purposes. This review discusses some of the concepts.

Affinity Labels↗
PubMed1989

Potent and selective inactivation of cysteine proteinases with N-peptidyl-O-acyl hydroxylamines.

A series of N-peptidyl-O-acyl hydroxylamines was synthesized and tested as inactivators of cysteine proteinases. Depending on the structure of the peptidyl residue of the inhibitors, rapid and complete irreversible inactivation of the lysosomal cathepsins, B, L and S, may be achieved. The most effective inhibitors display second-order rate constants of the inactivation in the range 10(5)-10(6) M-1.s-1. By contrast, the activity of the aminoendopeptidase cathepsin H is only negligibly affected by the N-terminal-protected peptidyl inhibitors.

Amino Acid Sequence↗
PubMed1989

Potent and selective inactivation of proteinases with N-peptidyl-O-acylhydroxylamines.

The reaction of seven N-peptidyl-O-acyl hydroxylamines with serine proteinases exhibiting different substrate specificity has been investigated. Depending on the structure of the peptidyl residue of the inhibitors, rapid and complete irreversible inactivation of the enzymes may be achieved. Enzyme-catalyzed turnover of inhibitor to products was detected during reaction of proline-specific endopeptidase with N-Boc-Ala-Pro-O-(4-nitrobenzoyl)hydroxylamine.

Endopeptidases↗
PubMed1989

Dipeptidyl peptidase IV in human T lymphocytes. Impaired induction of interleukin 2 and gamma interferon due to specific inhibition of dipeptidyl peptidase IV.

Specific inhibitors of the membrane-bound dipeptidyl peptidase IV (DP IV) and polyclonal antibodies against this enzyme were used to investigate the relationships between DP IV activity and the production and action of T cell-derived lymphokines. Production of interleukin 2 (IL-2) and gamma interferon by mitogen plus phorbol ester-stimulated mononuclear cells from human blood was found to be reduced in the presence of N-Ala-Pro-O-(nitrobenzoyl-)-hydroxylamine, epsilon-(4'-nitro) benzoxycarbonyl-Lys-Pro, and anti-(DP IV) immunoglobulin in a dose-dependent manner. Moreover, the proliferative response of mitogen-stimulated mononuclear cells to IL-2 is impaired in the presence of DP IV inhibitors. Therefore it is suggested that the membrane peptidase DP IV is involved in the induction and activation of cytokines controlling lymphocyte proliferation.

Cells, Cultured↗