| 词条 | Thermolysin |
| 释义 |
| Name = Thermolysin | EC_number = 3.4.24.27 | CAS_number = 9073-78-3 | IUBMB_EC_number = 3/4/24/27 | GO_code = | image = 3TMN.jpeg | width = | caption = Crystallographic structure of Bacillus thermoproteolyticus thermolysin.[1] }} Thermolysin ({{EC number|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). SynthesisLike 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] StructureThermolysin 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
References1. ^{{PDB|3TMN}}; {{cite journal |vauthors=Holden HM, Matthews BW | title = The binding of L-valyl-L-tryptophan to crystalline thermolysin illustrates the mode of interaction of a product of peptide hydrolysis | journal = J. Biol. Chem. | volume = 263 | issue = 7 | pages = 3256–60 |date=March 1988 | pmid = 3343246 | doi = | url = }} 2. ^{{cite journal | author=Endo, S. | title= Studies on protease produced by thermophilic bacteria | journal= J. Ferment. Technol. | year=1962 | volume=40 | pages=346–353}} 3. ^{{cite journal |vauthors=Tajima M, Urabe I, etal | title= Role of calcium ions in the thermostability of thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease | journal= Eur. J. Biochem. | year=1976 | volume=64 | issue=1 | pages=243–247 | pmid=819262 | doi=10.1111/j.1432-1033.1976.tb10293.x}} 4. ^{{cite journal | author=Trusek-Holownia A. | title= Synthesis of ZAlaPheOMe, the precursor of bitter dipeptide in the two-phase ethyl acetate-water system catalysed by thermolysin | journal= J. Biotechnol. | year=2003 | volume=102 | issue=2 | pages=153–163 | pmid=12697393 | doi=10.1016/S0168-1656(03)00024-5}} 5. ^{{cite journal |vauthors=Yasukawa K, Kusano M, Inouye K | title= A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli | journal= Protein Eng. Des. Sel. | year=2007 | volume=20 | issue=8 | pages=375–383| pmid=17616558 | doi=10.1093/protein/gzm031}} 6. ^{{cite book |vauthors=Inouye K, Kusano M, etal | title=Engineering, expression, purification, and production of recombinant thermolysin | journal=Biotechnol. Annu. Rev. | year=2007 | volume=13 | pages=43–64 | pmid=17875473 | doi=10.1016/S1387-2656(07)13003-9 | series=Biotechnology Annual Review | isbn=978-0-444-53032-5}} 7. ^{{cite journal |vauthors=Holmes MA, Matthews BW| title=Structure of thermolysin refined at 1.6 A resolution | journal=J. Mol. Biol. | year=1982 | volume=160 | issue=4 | pages=623–639 | pmid=7175940 | doi=10.1016/0022-2836(82)90319-9}} 8. ^{{cite journal |vauthors=Matthews BW, Weaver LH, Kester WR | title=The conformation of thermolysin | journal=J. Biol. Chem. | year=1974 | volume=249 | issue=24 | pages=8030–8044 | pmid=4214815}} 9. ^1 2 {{cite journal |vauthors=Eijsink VG, Veltman OR, etal | title=Structural determinants of the stability of thermolysin-like proteinases | journal=Nat. Struct. Biol. | year=1995 | volume=2 | issue=5 | pages=374–379 | pmid=7664094 | doi=10.1038/nsb0595-374}} 10. ^{{cite journal |vauthors=Dahlquist FW, Long JW, Bigbee WL | title=Role of Calcium in the thermal stability of thermolysin | journal=Biochemistry | year=1976 | volume=15 | issue=5 | pages=1103–1111 | pmid=814920 | doi=10.1021/bi00650a024}} 11. ^{{cite journal |last= Yagasaki |first= Makoto |author2=Hashimoto, Shin-ichi |date=November 2008 |title= Synthesis and application of dipeptides; current status and perspectives |journal= Applied Microbiology and Biotechnology |volume= 81 |issue= 1 |pages= 13–22 |pmid= 18795289 |doi= 10.1007/s00253-008-1590-3 }} 12. ^{{cite journal| title=Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp| journal=PLOS ONE| volume=7| issue=10| pages=e46147| doi=10.1371/journal.pone.0046147| pmid=23056252| pmc=3463568|year = 2012|last1 = Minde|first1 = David P.| last2=Maurice| first2=Madelon M.| last3=Rüdiger| first3=Stefan G. D.}} External links
3 : EC 3.4.24|Zinc proteins|Peptidase |
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