Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this work, the helix-forming residues in fragments of several proteins (ribonuclease, thermolysin, tendamistat and angiogenin) were identified by NOE and the helix proton shifts were measured as delta changes associated with helix-population increments driven by trifluoroethanol addition. When estimated in this way, a regular pattern of helix conformational shifts was clearly seen in the delta delta versus sequence profiles of all the peptides studied. The helix periodicity of the H alpha and H beta resonances was especially clear, an observation that earlier statistical studies of protein delta values failed to predict. Amide protons showed the largest helix shifts, but with a less-sharply defined periodic character. Aromatic residues considerably distorted the periodicity of the helix amide shifts in some peptides, as evidenced by the delta shifts of a RNase A fragment 1-15 analog in which the two aromatic residues were replaced by Ala. The relationship between helix periodicity and peptide amphiphatic character is discussed.
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PMID:Periodic properties of proton conformational shifts in isolated protein helices. An experimental study. 162 61

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

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

The flavoprotein ferredoxin-NADP+ reductase is inactivated and loses its ability to bind NADP+ during covalent modification of a lysine by 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) [Zanetti, G. (1976) Biochim. Biophys. Acta 445, 14-24]. The substrate NADP+ gives almost complete protection against inactivation and modification. These observations are extended in this report by the characterization of an octapeptide containing the dansyl-lysine which was isolated by high-performance liquid chromatography from tryptic digests of protein modified with radiolabeled reagent. The amount of this peptide was severely reduced in protein modified in the presence of NADP+. The sequence of the dansyl-peptide, only partially obtained by Edman degradation, was completed by analysis of the fragments resulting from thermolysin digestion of the purified tryptic dansyl-peptide. Thus, the octapeptide containing the essential lysine residue has the following sequence: H2N-Ser-Val-Ser-Leu-Cys-Val-Lys-Arg-COOH. A comparison with corresponding sequences of other known NADP+-dependent dehydrogenases is attempted.
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PMID:The NADP+-binding site of ferredoxin-NADP+ reductase. Sequence of the peptide containing the essential lysine residue. 391 22

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

The complete amino acid sequence of the DNA-binding histone-like protein (Hta) from Thermoplasma acidophilum has been established by sequence studies directly on the protein and on tryptic, chymotryptic, and thermolysin peptides derived from the protein. The sequence of the 89-residue form of HTa is: H2N-Val-Gly-Ile-Ser-Glu-Leu-Ser-Lys-Glu-Val-Ala-Lys-Lys-Ala-Asn-Thr-Thr-Gln-Lys -Val-Ala-Arg-Thr-Val-Ile-Lys-Ser-Phe-Leu-Asp-Glu-Ile-Val-Ser-Glu-Ala-Asn-Gly-Gl y-Gln-Lys-Ile-Asn-Leu-Ala-Gly-Phe-Gly-Ile-Phe-Glu-Arg-Arg-Thr-Gln-Gly-Pro-Arg-L ys-Ala-Arg-Asn-Pro-Gln-Thr-Lys-Lys-Val-Ile-Glu-Val-Pro-Ser-Lys-Lys-Lys-Phe-Val- Phe-Arg-Ala-Ser-Ser-Lys-Ile-Lys-Tyr-Gln-Gln-COOH The molecular weight calculated from the sequence is 9,934. Another form of HTa probably differs only by the presence of an additional residue (methionine) at the NH2 terminus (the calculated molecular weight of this form is 10,065). HTa resembles eukaryotic histones in several ways, including some sequence homology, HTa also shows sequence homology with the Escherichia coli DNA-binding proteins NS1 (or HU-1) and NS2 (or HU-2).
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PMID:A histone-like protein (HTa) from Thermoplasma acidophilum. II. Complete amino acid sequence. 700 26

Conformational dynamics are thought to be a prerequisite for the catalytic activity of enzymes. However, the exact relationship between structural fluctuations and function is not well understood. In this work hydrogen/deuterium exchange (HDX) and electrospray ionization mass spectrometry (ESI-MS) are used for exploring the conformational dynamics of thermolysin. Amide HDX reflects the internal mobility of proteins; regions that undergo frequent unfolding-refolding show faster exchange than segments that are highly stable. Thermolysin is a zinc protease with an active site that is located between two lobes. Substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. HDX/ESI-MS with proteolytic peptide mapping in the absence of substrate shows that elements in the periphery of the two lobes are most mobile. A comparison with previous X-ray data suggests that these peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The presence of both rigid and moderately flexible elements in the active site may reflect a carefully tuned balance that is required for function. Interestingly, the HDX behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme. This result is consistent with the view that catalytically relevant motions preexist in the resting state and that enzyme function can only be performed within the limitations given by the intrinsic dynamics of the protein. The data presented in this work indicate the prevalence of stochastic elements in the function of thermolysin, rather than supporting a deterministic mechanism.
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PMID:Conformational dynamics of free and catalytically active thermolysin are indistinguishable by hydrogen/deuterium exchange mass spectrometry. 1849