UNIPROT:P15088 - FACTA Search

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Query: UNIPROT:P15088 (mast cell)
14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

L-Leucinthiol, a synthetic derivative of mercaptoethylamine with a hydrophobic side chain, was recently reported to be a potent inhibitor of microsomal aminopeptidase. The structural features necessary for interaction of mercaptoamines with this enzyme have now been explored more systematically. Optimal binding requires a primary amine linked to the mercapto group via two carbon atoms. Only a substituent with L-configuration at the 1 position increased the affinity toward the enzyme. The high degree of specificity and other evidence suggest that the mode of binding of these inhibitors is similar to that of substrates. Comparison of leucinthiol with other amino compounds suggest that the mercapto group makes a much greater contribution to the binding than the hydrophobic side chain. L-Leucinthiol is fairly specific for aminopeptidase although some inhibition of thermolysin and carboxypeptidase A is observed.
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PMID:Structural requirements for specific inhibition of microsomal aminopeptidase by mercaptoamines. 400 71

Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]glycine is a highly potent inhibitor (Ki = 3.5 nM) of "enkephalinase," a metalloendopeptidase cleaving the Gly-Phe bond (positions 3 and 4) of enkephalins in brain tissue. In accordance with this property, thiorphan displays antinociceptive activity after systemic administration. However, thiorphan also inhibits to a lesser extent (Ki = 140 nM) the widely distributed angiotensin-converting enzyme, a carboxydipeptidase implicated in blood pressure regulation. Therefore, in view of an eventual clinical use of enkephalinase inhibitors, it was very important to develop fully specific compounds. Such derivatives were obtained taking into account that N-methylation of the ultimate amide bond of dipeptides strongly decreases enkephalinase affinity without affecting angiotension-converting enzyme recognition, whereas retro-inversion of the amide bond leads to the inverse effect. Thus, the retro-inverso dipeptide (R)-H2N-CH(CH2 phi)-NHCO-CH2-CO2H exhibits an inhibitory potency on enkephalinase (IC50 approximately equal to 12 muM) close to that of the natural dipeptide L-Phe-Gly (IC50 approximately equal to 3 muM). This result shows the topological analogy between the crucial components involved in enkephalinase recognition both in active dipeptides and structurally related retro-inverso isomers. Taking into account these observations, retro-thiorphan, (R,S)-HS-CH2-CH-(CH2 phi)-NHCO-CH2-COOH, was prepared. As compared to thiorphan, the retro isomer is 50% as potent (Ki = 6 nM) on enkephalinase but displays a drastic loss of potency on angiotension-converting enzyme (IC50 greater than 10,000 nM). This specificity was interpreted as a consequence of differences in the stereochemical constraints involving enzyme-inhibitor hydrogen bonding. This hypothesis is supported by reported crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. As expected, retro-thiorphan exhibits about the same analgesic potency as thiorphan on the hot plate and writhing tests in mice. Therefore, the topological concept of retro-inverso isomers could be extended to other enkephalinase inhibitors, allowing the design of potent and highly selective compounds occurring as new classes of analgesic and psychoactive agents.
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PMID:Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan. 630 95

Despite the similarities in their mechanism of action, the structural requirements for selective interaction with angiotensin-converting enzyme or enkephalinase are different. Inhibitory potency of a series of new mercaptoalkanoyl amino acids were determined on pure angiotensin-converting enzyme (EC 3.4.15.1) from porcine plasma and on neutral metalloendopeptidase (EC 3.4.24.11) purified from rat brain. This latter enzyme, first designated as enkephalinase, seems to be synaptically involved in the degradation of enkephalins. All tested compounds, whose design was based on the classical active-site model of metallopeptidases, are reversible and competitive inhibitors of both enzymes. Owing to the remarkable similarity in the general topology of metallopeptidases, the differences in optimal binding requirements to enkephalinase and angiotensin-converting enzyme were interpreted from crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. The large size of the S'1 subsite of enkephalinase allows efficient binding (Ki approximately equal to 2-30 nM) of aromatic and bulky hydrophobic residues such as a cyclohexyl ring. In contrast, a methyl group in position P'1 favors inhibitory potency against angiotensin-converting enzyme while a cyclohexyl ring leads to a complete loss of activity. This feature could mean that optimal binding of the Zn atom present in the catalytic site is a more stringent requirement in angiotensin-converting enzyme than in enkephalinase. An increase in the size of the P'2 component of thiol inhibitors potentiates the affinity for angiotensin-converting enzyme without a significant change on enkephalinase. Finally, methylation of the ultimate amide bond of inhibitors produces a 30-fold decrease in potency towards enkephalinase but does not affect the binding of angiotensin-converting enzyme. These findings allow a rational design of selective inhibitors of enkephalinase, an essential prerequisite for their possible clinical use as new analgesic and psycho-active agents.
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PMID:Differences in the structural requirements for selective interaction with neutral metalloendopeptidase (enkephalinase) or angiotensin-converting enzyme. Molecular investigation by use of new thiol inhibitors. 632 Nov 77

The mode of binding of the specific thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan (KI approximately 5 X 10(-8) M) [Maycock, A. L., DeSousa, D. M., Payne, L. G., ten Broeke, J., Wu, M. T., & Patchett, A. A. (1981) Biochem. Biophys. Res. Commun. 102, 963-969] has been determined by X-ray crystallography and refined to an R value of 17.1% at 1.9-A resolution. The inhibitor binds to thermolysin with both oxygens of the N-carboxymethyl group liganded to the zinc to give overall pentacoordination of the metal. The bidentate ligation of the inhibitor differs from the monodentate binding seen previously for carboxylate-zinc interactions in thermolysin and is closer to the bidentate geometry observed for the binding of hydroxamates [Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912-6920]. The geometry of the inhibitor and its interactions with the protein have a number of elements in common with the presumed transition state formed during peptide hydrolysis. The observed zinc ligation supports the previous suggestion that a pentacoordinate intermediate participates in the mechanism of catalysis. However, the alpha-amino nitrogen of the inhibitor is close to Glu-143, suggesting that this residue might accept a proton from an attacking water molecule (as proposed before) and subsequently donate this proton to the leaving nitrogen. By analogy with thermolysin, it is proposed that a related mechanism should be considered for peptide cleavage by carboxypeptidase A.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of N-carboxymethyl dipeptide inhibitors to thermolysin determined by X-ray crystallography: a novel class of transition-state analogues for zinc peptidases. 639 81

Reexamination of the molecular mass and the amino acid composition of Serratia protease revealed the presence of 1 mol of methionine per mol of protein (about 46K daltons), and this was confirmed by BrCN cleavage followed by separation of the two fragments. The sole methionine residue was located near the middle region of the molecule. The amino(N)-terminal sequence was determined by Edman degradation of the protein and studies of several proteolytic peptides, establishing a sequence of 18 residues with a heterogeneous N-terminus. The carboxyl(C)-terminal sequence was determined by carboxypeptidase A digestion and tritium-labeling of the citraconylated C-terminal half segment to be -Phe-Ile-Val. The sequences of a total of 53 residues containing the methionine residue and a total of 38 residues containing two histidine residues were established by the application of various conventional methods to a BrCN peptide and several proteolytic peptides. The segment containing the histidine residues was homologous with that containing the two histidine residues chelating the zinc atom of thermolysin. The 38-residue segment may be directly connected to the 53-residue segment.
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PMID:Serratia protease. Amino acid sequences of both termini, the 53 residues in the middle region containing the sole methionine residue, and a probable zinc-binding region. 639 98

The 22076-Mr Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces abuls G effectively catalyses the transfer of the N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl electrophilic group of the standard tripeptide substrate N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl-D-alanine to water. It also performs a weak beta-lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[14C]-acetamide, and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2+/serine-containing beta-lactamases.
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PMID:The complete amino acid sequence of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of streptomyces albus G. 682 89

The mode of binding of the irreversible thermolysin inhibitor ClCH2CO-DL-(N-OH)Leu-OCH3 [Rasnick, D., & Powers, J.C. (1978) Biochemistry 17, 4363-4369] has been determined by X-ray crystallography at a resolution of 2.3 A and the structure of the covalent complex refined to give a crystallographic residual of 17.0%. This is the first such structural study of an active-site-directed covalent complex of a zinc protease. As anticipated by Rasnick and Powers, the inhibitor alkylates Glu-143 in the thermolysin active site, and the hydroxamic acid moiety coordinates the zinc ion. The formation of the covalent complex is associated with a significant shift in a segment of the polypeptide backbone in the vicinity of the active site. This conformational adjustment appears to be necessary to relieve steric hindrance which would otherwise prevent alkylation of Glu-143. It is suggested that this steric hindrance, which occurs for thermolysin but would not be expected for carboxypeptidase A, accounts for the previously inexplicable difference in reactivity of these two metalloproteases toward N-haloacetyl amino acids. The relevance of this steric hindrance to the mechanism of catalysis is discussed. In agreement with previous results [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506-2516], it appears that steric hindrance prevents the direct attack of Glu-143 on the carbonyl carbon of an extended substrate, therefore ruling out the anhydride pathway in thermolysin-catalyzed hydrolysis of polypeptide substrates and their ester analogues.
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PMID:Structural analysis of the inhibition of thermolysin by an active-site-directed irreversible inhibitor. 683 Jul 61

The assignment of five disulfide bonds in the alpha subunit of human chorionic gonadotropin (hCG) using partial reduction and S-[14C]carboxymethylation has been reported earlier (Mise, T., and Bahl, O. P. (1980) J. Biol. Chem. 255, 8516-8522). Employing a similar approach, we have determined the locations of six disulfide bonds in hCG-beta. Two partially reduced and S-[14C]carboxymethylated hCG-beta derivatives, DS1.4-hCG-beta and DS3.4-hCG-beta in which on the average 1.4 and 3.4 disulfide bonds were modified, respectively, were prepared. The 14C-labeled derivatives were then completely reduced and S-carboxymethylated with nonradioactive iodoacetic acid and subjected to hydrolysis with trypsin. The radioactive peptides were purified by gel filtration and high voltage paper electrophoresis. The tryptic peptides containing two or more S-[14C]carboxymethylcysteines were further degraded using various proteolytic enzymes such as thermolysin, carboxypeptidase A and Y, cathespin C, and subtilisin to obtain individual S-[14C]carboxymethylcysteine-containing peptides. From the specific radioactivities of S-[14C]carboxymethylcysteines in DS3.4-hCG-beta, four out of six disulfide bonds, 9-90, 26-110, 34-88, and 93-100 were assigned. Similar data from DS1.4-hCG-beta gave the locations of the other two disulfide bonds, 23-72 and 38-57, while confirming the locations of four disulfide bonds derived from the radioactivity distribution in DS3.4-hCG-beta. Thus, all six disulfide bonds in hCG-beta have been located. The results of controlled reduction and S-[14C]alkylation also indicate that disulfide bond 93-100 is the most reactive, followed by disulfide bond 26-110, and that the least reactive among all is 34-88.
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PMID:Assignment of disulfide bonds in the beta subunit of human chorionic gonadotropin. 724 Feb 31

Complexes of cobalt(II) and zinc(II) which involve monodentate coordination of two alkyl carboxylate and two imidazole ligands in a slightly distorted tetrahedral fashion have visible and magnetic circular dichroism spectra remarkably similar to the cobalt(II)-substituted proteolytic enzymes thermolysin and carboxypeptidase A. Single crystal x-ray structure determinations on [Co(C2H5COO)2Im2], Im = imidazole, and its zinc counterpart reveal only minor structural differences between the cobalt and zinc species. Electron paramagnetic resonance spectra of cobalt(II) doped into zinc(II) complexes with known structures demonstrate the extreme sensitivity of the g-values to minor structural differences.
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PMID:Structural and electronic mimics of the active site of cobalt(II)-substituted zinc metalloenzymes. 744 Dec 45

Within the past few years, members of the hedgehog (hh) family of secreted signalling proteins have emerged as the primary signals generated by certain embryonic patterning centres. In vertebrate embryos, for example, sonic hedgehog expression in the notochord appears to be responsible for the local and long-range induction of ventral cell types within the neural tube and somites (reviewed in refs 1, 2). Protein products encoded by hh family members are synthesized as precursors that undergo autoprocessing to generate an amino-terminal domain that appears to be responsible for both local and long-range signalling activities, and a carboxy-terminal domain that contains the autoprocessing activity. As part of an effort to understand how hh family members participate in cell-to-cell signalling, we have determined and report here the crystal structure at 1.7 A of the amino-terminal domain of murine Sonic hedgehog (Shh-N). The structure revealed a tetrahedrally coordinated zinc ion that appears to be structurally analogous to the zinc coordination sites of zinc hydrolases, such as thermolysin and carboxypeptidase A. This previously unsuspected catalytic site represents a distinct activity from the autoprocessing activity that resides in the carboxy-terminal domain.
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PMID:A potential catalytic site revealed by the 1.7-A crystal structure of the amino-terminal signalling domain of Sonic hedgehog. 747 29

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