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).
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]
