Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.27 (thermolysin)
1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.
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PMID:Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin. 235 Jan 81

Structure-function relations of the colicin E1 ion channel were studied through the effects of mutations in the 35-residue hydrophobic region of the channel polypeptide and neighboring residues in the channel domain. Mutation of neutral residues threonine 501 and glycine 502 to a more polar or charged glutamic acid generated a protein whose channel conductance properties in each case had a decreased selectivity for anions. There was no significant effect on ion selectivity caused by mutations that changed residue charge outside the hydrophobic domain at the neighboring aspartic acid 509 or at glycine 439. The Thr501----Glu and Gly502----Glu mutants possessed lower cytotoxic and in vitro activity. An altered thermolysin cleavage pattern and a greater binding to membrane vesicles at pH greater than 4.5 of the Gly502----Glu mutant indicated greater exposure of its COOH-terminal hydrophobic domain in solution. It is concluded that the hydrophobic nature of threonine 501 and glycine 502 is important in the structure of the channel lumen and the soluble colicin. Altering proline 462, a residue conserved in five sequenced channel-forming colicins, had no significant effect on channel properties. These conclusions are discussed in the context of sequence-structure-function concepts for channel proteins.
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PMID:Decrease of anion selectivity caused by mutation of Thr501 and Gly502 to Glu in the hydrophobic domain of the colicin E1 channel. 256 69

A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.
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PMID:Sequencing of an active-site peptide of angiotensin I-converting enzyme containing an essential glutamic acid residue. 285 12

Photoaffinity labeling with native NAD, a method employed earlier with diphtheria toxin (DT), was used to identify an active site residue of Pseudomonas aeruginosa exotoxin A (ETA). An enzymically active fragment (Mr 27,000), derived by partial digestion of ETA with thermolysin, was irradiated with ultraviolet light (254 nm) in the presence of various radiolabeled preparations of NAD. Label from the nicotinamide moiety was efficiently transferred to the protein (maximally 0.79 mol/mol), and the label was exclusively located at position 553. This position, like that photolabeled in DT (position 148), corresponds to glutamic acid in the native protein. Chromatographically identical photo-products were generated at these positions in the two toxins. Glu-553 lies in a cleft in domain III that is believed to represent the active site of ETA, and other evidence supports the notion that Glu-553 of ETA and Glu-148 of DT are directly involved in catalysis. When Glu-553 of ETA was aligned with Glu-148 of DT, we found similarities of local primary structure not detected earlier. These results suggest that the catalytically active domains of ETA and DT may be evolutionarily related, and they provide information that should prove useful for preparing vaccines against ETA by recombinant DNA methods.
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PMID:Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. 288 23

A glutamic acid residue at the active-site of bovine lung angiotensin I-converting enzyme was esterified with p-[N,N-bis-(chloroethyl)amino]phenylbutyryl-L-[U-14]-Proline (chlorambucyl-L-[U-14C]-L-Proline), an affinity label for this enzyme. The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase HPLC contained the bound radiolabel. This active-site peptide (Mr approximately 16,000) was digested with trypsin, and the labeled peptide (T-2) was further degraded with thermolysin. The enzyme digest peptides were also resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained after thermolysin digestion (Th-1, Mr 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu. The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of PTH-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]-Proline which confirms our earlier findings. The sequence that we determined is homologous in five residues with the corresponding sequences of carboxypeptidase A and B, two other mammalian zinc-proteases. There is little sequence homology with thermolysin, a bacterial zinc-protease that also contains an essential active-site glutamic acid residue.
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PMID:Isolation and sequencing of an active-site peptide from angiotensin I-converting enzyme. 302 71

Storage proteins of the albumin solubility fraction from seeds of Bertholletia excelsa H.B.K. were separated by reversed-phase high-performance liquid chromatography and their primary structures were determined by gas-phase sequencing on intact polypeptides and on the overlapping tryptic and thermolysin peptides. The 2S storage proteins consist of two subunits linked by disulphide bridges. The large subunit (8.5 kDa) is expressed in at least six different isoforms while the small subunit (3.6 kDa) consists of only one form. These proteins are extremely rich in glutamine, glutamic acid, arginine and the sulphur-containing amino acids cysteine and methionine. One of the variants even contains a sequence of six methionine residues in a row. Comparison with known sequences of 2S proteins of other dicotyledonous plants shows limited but distinct sequence homology. In particular, the positions of the cysteine residues relative to each other appear to be completely conserved, suggesting that tertiary structure constraints imposed by disulphide bridges dominate sequence conservation. It has been proposed that the two subunits of a related protein (the Brassica napus storage protein) is cleaved from a precursor polypeptide [Crouch, M. L., Tenbarge, K. M., Simon, A. E. & Ferl, R. (1983) J. Mol. Appl. Genet. 2,273-283]. The amino acid sequence homology of the Brazil nut protein with the former suggests that a similar protein processing event could occur.
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PMID:The amino-acid sequence of the 2S sulphur-rich proteins from seeds of Brazil nut (Bertholletia excelsa H.B.K.). 375 80

Tritium-containing affinity-labelling derivatives of phalloidin, an alkylating iodoacetyl compound (EAL) and a photolabile, carbene generating diazirine (PAL), have been reacted with rabbit muscle actin, the former after protection of thiol groups with N-ethylmaleimide. Labelled peptides generated by tryptic and/or thermolysin digestion were isolated by paper peptide mapping and characterized by determination of their amino acid sequences. EAL binds to methionine-119 and methionine-355; PAL binds to glutamic acid-117. These residues are located in regions with extremely conserved amino acid sequences. The cleft between the two domains of the actin monomer is suggested as the possible binding site for phalloidin.
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PMID:The phalloidin binding site of F-actin. 406 95

An endogenous inhibitor of calcium-activated neutral protease (CANP), which was isolated from rabbit skeletal muscle under mild conditions, comprised high- and low-molecular-weight components. The latter (LMW-inhibitor; Mr=50,000) was purified to homogeneity by means of chromatography on DEAE-cellulose and phenyl-Sepharose CL-4B and chromatofocusing. The purified inhibitor is a protein composed of two polypeptide chains with molecular weights of 26,000 and 24,000 daltons. It contains large amounts of glutamic acid, alanine, and serine, and small amounts of aromatic amino acids. It was specific for CANPs having low (m-type) and high (mu-type) Ca2+-sensitivity, had no effect on any other protease examined (trypsin, alpha-chymotrypsin, bromelain, ficin, papain, thermolysin, etc.), and inhibited rabbit mCANP more effectively than rabbit muCANP or chicken mCANP. It was demonstrated that the inhibition is due to the formation of a stoichiometric complex between two molecules of rabbit mCANP and one inhibitor molecule.
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PMID:Purification and characterization of an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle: purification of 50,000-dalton inhibitor. 609 76

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic-AMP-dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N-terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr = 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB2 (apparent Mr = 24 000) at the C-terminal end of the molecule. The sequence of the N-terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5, 1a and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N-terminus of CB2 respectively. Site 1a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites 1a and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N-terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C-terminal to site 3c while chymotrypsin cleaves N-terminal to site 3a has formed the basis of a simple procedure for determining the state of phosphorylation of the seven serine residues in vivo [Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem. 124, 47-55].
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PMID:Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain. 680 97

A cDNA coding for the E isoform of alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) was isolated by oligonucleotide hybridization following immunochemical screening of the rabbit liver cDNA library. The deduced amino acid sequence of the E isoform showed 96.4% identity in 413 residues of the F and S-1 isoforms of rabbit alpha-1-antiproteinase. The N-terminal half of the amino acid residues of the three isoforms was almost identical, but the putative reactive-site loop structure (P8-P'8) was significantly different in the various forms, the P1 site of the E form being glutamic acid. Interaction of the recombinant E form with the various proteinases was investigated by SDS/PAGE, followed by immunoblot analysis. The recombinant protein and trypsin formed a 62 kDa equimolar complex, which gradually became graded to the 37 kDa fragment through several intermediates. The E form also formed a complex of a similar size with elastase and became degraded to the 31 kDa fragment. Several proteinases which cleaved the E form without forming a detectable complex on SDS/PAGE are chymotrypsin, protease V8, pancreas kallikrein, thermolysin, papain and ficin. Other proteinases, with a stringent substrate specificity, such as thrombin, factor Xa, plasmin, plasma kallikrein and cathepsin G, did not attack the E form. Unlike the F and S-1 forms of rabbit plasma alpha-1-antiproteinase, the recombinant E form did not inhibit the amidolytic and proteolytic activities of trypsin. Neither elastase nor protease V8 was inhibited by the E form. Thus the change in the amino acid residues in the reactive-site loop, probably in the P1 site, is responsible for the loss of inhibitory activity of rabbit alpha-1-antiproteinase E. The novel character of the E form could provide a new insight into the interaction of serpin and proteinases.
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PMID:Rabbit alpha-1-antiproteinase E: a novel recombinant serpin which does not inhibit proteinases. 773 71


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