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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2016/02/25 04:22:31」(JST)
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| Thermolysin |
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Crystallographic structure of Bacillus thermoproteolyticus thermolysin.[1]
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| Identifiers |
| EC number |
3.4.24.27 |
| CAS number |
9073-78-3 |
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IntEnz view |
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BRENDA entry |
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KEGG entry |
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RCSB PDB PDBe PDBsum |
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proteins |
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Thermolysin (EC 3.4.24.27, Bacillus thermoproteolyticus neutral proteinase, thermoase, thermoase Y10, TLN) is a thermostable neutral metalloproteinase enzyme produced by the Gram-positive bacteria Bacillus thermoproteolyticus.[2] It requires one zinc ion for enzyme activity and four calcium ions for structural stability.[3] Thermolysin specifically catalyzes the hydrolysis of peptide bonds containing hydrophobic amino acids. However thermolysin is also widely used for peptide bond formation through the reverse reaction of hydrolysis.[4] Thermolysin is the most stable member of a family of metalloproteinases produced by various Bacillus species. These enzymes are also termed 'neutral' proteinases or thermolysin -like proteinases (TLPs).
Contents
- 1 Synthesis
- 2 Structure
- 3 Applications
- 4 References
- 5 External links
Synthesis
Like all bacterial extracellular proteases thermolysin is first synthesised by the bacterium as a pre-proenzyme.[5] Thermolysin is synthesized as a pre-proenzyme consisting of a signal peptide 28 amino acids long, a pro-peptide 204 amino acids long and the mature enzyme itself 316 amino acids in length. The signal peptide acts as a signal for translocation of pre-prothermolysin to the bacterial cytoplasmic membrane. In the periplasm pre-prothermolysin is then processed into prothermolysin by a signal peptidase. The prosequence then acts as a molecular chaperone and leads to autocleavage of the peptide bond linking pro and mature sequences. The mature protein is then secreted into the extracellular medium.[6]
Structure
Thermolysin has a molecular weight of 34,600 Da. Its overall structure consists of two roughly spherical domains with a deep cleft running across the middle of the molecule separating the two domains. The secondary structure of each domain is quite different, the N-terminal domain consists of mostly beta pleated sheet, while the C-terminal domain is mostly alpha helical in structure. These two domains are connected by a central alpha helix, spanning amino acids 137-151.[7]
In contrast to many proteins that undergo conformational changes upon heating and denaturation, thermolysin does not undergo any major conformational changes until at least 70 °C.[8] The thermal stability of members of the TLP family is measured in terms of a T50 temperature. At this temperature incubation for 30 minutes reduces the enzymes activity by half. Thermolysin has a T50 value of 86.9 °C, making it the most thermo stable member of the TLP family.[9] Studies on the contribution of calcium to thermolysin stability have shown that upon thermal inactivation a single calcium ion is released from the molecule.[10] Preventing this calcium from originally binding to the molecule by mutation of its binding site, reduced thermolysin stability by 7 °C. However, while calcium binding makes a significant contribution to stabilising thermolysin, more crucial to stability is a small cluster of N-terminal domain amino acids located at the proteins surface.[9] In particular a phenylalanine (F) at amino acid position 63 and a proline (P) at amino acid position 69 contribute significantly to thermolysin stability. Changing these amino acids to threonine (T) and alanine (A) respectively in a less stable thermolysin-like proteinase produced by Bacillus stearothermophillus (TLP-ste), results in individual reductions in stability of 7 °C (F63→T) and 6.3 °C (P69→A) and when combined a reduction in stability of 12.3 °C.[9]
Applications
- In the synthesis of aspartame, less bitter-tasting byproduct is produced when the reaction is catalyzed by thermolysin.[11]
- Determining protein stability in cell lysate using the fast parallel proteolysis (FASTpp) assay.[12]
References
- ^ PDB: 3TMN; Holden HM, Matthews BW (March 1988). "The binding of L-valyl-L-tryptophan to crystalline thermolysin illustrates the mode of interaction of a product of peptide hydrolysis". J. Biol. Chem. 263 (7): 3256–60. PMID 3343246.
- ^ Endo, S. (1962). "Studies on protease produced by thermophilic bacteria". J. Ferment. Technol. 40: 346–353.
- ^ Tajima M, Urabe I, et al. (1976). "Role of calcium ions in the thermostability of thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease". Eur. J. Biochem. 64 (1): 243–247. doi:10.1111/j.1432-1033.1976.tb10293.x. PMID 819262.
- ^ Trusek-Holownia A. (2003). "Synthesis of ZAlaPheOMe, the precursor of bitter dipeptide in the two-phase ethyl acetate-water system catalysed by thermolysin". J. Biotechnol. 102 (2): 153–163. doi:10.1016/S0168-1656(03)00024-5. PMID 12697393.
- ^ Yasukawa K, Kusano M, Inouye K. (2007). "A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli". Protein Eng. Des. Sel. 20 (8): 375–383. doi:10.1093/protein/gzm031. PMID 17616558.
- ^ Inouye K, Kusano M, et al. (2007). "Engineering, expression, purification, and production of recombinant thermolysin". Biotechnol. Annu. Rev. Biotechnology Annual Review 13: 43–64. doi:10.1016/S1387-2656(07)13003-9. ISBN 978-0-444-53032-5. PMID 17875473.
- ^ Holmes MA and Matthews BW. (1982). "Structure of thermolysin refined at 1.6 A resolution". J. Mol. Biol. 160 (4): 623–639. doi:10.1016/0022-2836(82)90319-9. PMID 7175940.
- ^ Matthews BW, Weaver LH and Kester WR. (1974). "The conformation of thermolysin". J. Biol. Chem. 249 (24): 8030–8044. PMID 4214815.
- ^ a b c Eijsink VG, Veltman OR, et al. (1995). "Structural determinants of the stability of thermolysin-like proteinases". Nat. Struct. Biol. 2 (5): 374–379. doi:10.1038/nsb0595-374. PMID 7664094.
- ^ Dahlquist FW, Long JW and Bigbee WL (1976). "Role of Calcium in the thermal stability of thermolysin". Biochemistry 15 (5): 1103–1111. doi:10.1021/bi00650a024. PMID 814920.
- ^ Yagasaki, Makoto; Hashimoto, Shin-ichi (November 2008). "Synthesis and application of dipeptides; current status and perspectives". Applied Microbiology and Biotechnology 81 (1): 13–22. doi:10.1007/s00253-008-1590-3. PMID 18795289.
- ^ "Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp".
External links
- The MEROPS online database for peptidases and their inhibitors: M04.001
- Thermolysin at the US National Library of Medicine Medical Subject Headings (MeSH)
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Proteases: metalloendopeptidases (EC 3.4.24)
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| ADAM proteins |
- Alpha secretases
- ADAM9
- ADAM10
- ADAM17
- ADAM19
- ADAM2
- ADAM7
- ADAM8
- ADAM11
- ADAM12
- ADAM15
- ADAM18
- ADAM22
- ADAM23
- ADAM28
- ADAM33
- ADAMTS1
- ADAMTS2
- ADAMTS3
- ADAMTS4
- ADAMTS5
- ADAMTS8
- ADAMTS9
- ADAMTS10
- ADAMTS12
- ADAMTS13
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| Matrix metalloproteinases |
- Collagenases
- Gelatinases
- MMP3
- MMP7
- MMP10
- MMP11
- MMP12
- MMP13
- MMP14
- MMP15
- MMP16
- MMP17
- MMP19
- MMP20
- MMP21
- MMP23A
- MMP23B
- MMP24
- MMP25
- MMP26
- MMP27
- MMP28
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| Other |
- Neprilysin
- Procollagen peptidase
- Thermolysin
- Pregnancy-associated plasma protein A
- Bone morphogenetic protein 1
- Lysostaphin
- Insulin-degrading enzyme
- ZMPSTE24
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Proteins: enzymes
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| Activity |
- Active site
- Binding site
- Catalytic triad
- Oxyanion hole
- Enzyme promiscuity
- Catalytically perfect enzyme
- Coenzyme
- Cofactor
- Enzyme catalysis
- Enzyme kinetics
- Lineweaver–Burk plot
- Michaelis–Menten kinetics
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| Regulation |
- Allosteric regulation
- Cooperativity
- Enzyme inhibitor
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| Classification |
- EC number
- Enzyme superfamily
- Enzyme family
- List of enzymes
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| Types |
- EC1 Oxidoreductases(list)
- EC2 Transferases(list)
- EC3 Hydrolases(list)
- EC4 Lyases(list)
- EC5 Isomerases(list)
- EC6 Ligases(list)
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English Journal
- Determining protease substrate selectivity and inhibition by label-free supramolecular tandem enzyme assays.
- Ghale G, Ramalingam V, Urbach AR, Nau WM.SourceSchool of Engineering and Science, Jacobs University Bremen , Campus Ring 1, D-28759 Bremen, Germany.
- Journal of the American Chemical Society.2011 May 18;133(19):7528-35. Epub 2011 Apr 22.
- An analytical method has been developed for the continuous monitoring of protease activity on unlabeled peptides in real time by fluorescence spectroscopy. The assay is enabled by a reporter pair comprising the macrocycle cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO). CB7 functi
- PMID 21513303
Japanese Journal
- Susceptibility to proteases of anti-Tn-antigen MLS128 binding glycoproteins expressed in human colon cancer cells
- , , , , , , ,
- BioScience Trends 9(1), 49-55, 2015
- … It must contain multiple cleavage sites for trypsin and thermolysin since these proteases digested 110 kDa GP to MLS128-undetectable small fragments. …
- NAID 130005054681
- Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability.
- Kojima Kenji,Nakata Hiroki,Inouye Kuniyo
- Biochimica et biophysica acta -Proteins and Proteomics 1844(2), 330-338, 2014-02
- … Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1-157) and α-rich C-terminal (residues 158-316) domains. … Expression of thermolysin variants truncated from the C-terminus was examined in E. … The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. …
- NAID 120005359199
- Effects of Conversion of the Zinc-Binding Motif Sequence of Thermolysin, HEXXH, to That of Dipeptidyl Peptidase III, HEXXXH, on the Activity and Stability of Thermolysin
- MENACH Evans,HASHIDA Yasuhiko,YASUKAWA Kiyoshi,INOUYE Kuniyo
- Bioscience, biotechnology, and biochemistry 77(9), 1901-1906, 2013-09-23
- NAID 10031202742
Related Links
- サーモリシン は耐熱性のメタロプロテイナーゼです。タンパク質分解に抵抗性を示すタンパク質の消化を改善するために変性剤の代わりに高い消化温度が用いられる場合があります。サーモリシンはロイシン、フェニルアラニン ...
- Thermolysin 販売元 和光純薬工業(株) 販売元コード 201-08331,207-08333 製造元 製造元コード CAS.NO 9073-78-3 分子式 分子量 37,500 保存条件 冷蔵 (氷冷輸送) 適用法規 危険有害性 等級 生化学用 for Biochemistry EC.NO 3.4 ...
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