Thermolysin
- UniProt: P00800
Thermolysin is a thermostable enzyme, which belongs to the group of metallopeptidases. It is secreted by the thermophilic bacterium Bacillus thermoproteolyticus. Endoprotease as it catalyzes the cleavage of peptide bonds; doing, hydrolysis was performed on the amino side by large, hydrophobic residues ( for example, by leucine, isoleucine or phenylalanine) within the polypeptide.
The enzyme transfers the EC Number EC 3.4.24.27.
General
Thermolysin belongs to the zinc -containing metalloproteases and is one of the most studied enzymes of this superfamily. The three-dimensional structure was elucidated in 1972 by Matthews and co-workers at a resolution of 2.3 Å, 1.6 Å resolution now been reached.
Structure
Thermolysin has a molar mass of 34.6 kDa. The enzyme has an N-terminal and a C- terminal domain, which form a column for its active center. The N-terminal domain is composed mainly of β -sheets, the more stable C-terminal domain of α - helices.
Thermolysin requires a zinc ion and four calcium ions. The former is essential for enzyme activity, but hardly contributes to the thermal stability. The zinc ion is found in the active site. The thermal stability is mediated by the calcium ions. The calcium ions to stabilize the structural integrity and to protect the surface of the enzyme from the projecting loops ( loops ) in front of autoproteolysis.
The protein has no disulfide bridges.
Chemical Properties
The endoprotease is thermostable, their function optimum is 65-70 ° C at pH 8.0. At 80 ° C its structural half -life of one hour. A Hitzedenaturation of thermolysin is, however, rapidly and irreversibly. This is done by unfolding and autoproteolysis exposed sequences.
Catalytic mechanism
Although many available data on crystal structure analysis, and mutagenesis studies of the reaction kinetics, the exact catalytic mechanism is open to debate. Using computer simulations of crystallographic data but applies to the following reaction mechanism as the most likely.
In the native state, the zinc ion is tetrahedrally coordinated ago. In this case, the metal ion is complexed to three amino acid residues of the enzyme ( histidine 142 (His 142), histidine 146 (His 146) and glutamic acid 166 (Glu 166) ) and a water molecule ( see also figure below). The latter is common to all zinc metalloproteins.
After binding of the peptide to be hydrolyzed (step 1 in the figure) the zinc ion is pentacoordinated ago. At the same time, the water molecule is placed 143 (Glu 143 ) in the vicinity of glutamate and strongly polarized by this and the positively charged zinc atom. This has the consequence that the nucleophilicity of the water molecule is large enough to attack the peptide bonds.
The nucleophilic attack (step 2) is performed on the peptide binding, is a proton of water through the Glu 143 transferred to the nitrogen. The carbon atom of a peptide bond is now available tetrahedral. His 231 and tyrosine ( Tyr) 157 help to stabilize the carbonyl oxygen. Finally, the cleavage of the peptide bond occurs, while the second originating from the water proton via Glu 143 is transferred to the nitrogen atom.
The amine product leaving the active site as a protonated form ( step 3). The Carboxylprodukt is replaced by a new water molecule (step 4), so that the zinc atom is tetrahedrally coordinated again. A new reaction mechanism can begin.
The Glu -143 residue formed with the two histidines His 142 and 146 is a so-called matching sequence motif, the motif HEXXH. This motif is highly conserved in all representatives of the Zinkendoproteasen with a zinc atom.
Inhibitors
A natural inhibitor of thermolysin is Phosphoramidon from Streptomyces tanashiensis. The currently most effective inhibitor is the artificial ZFpLA ( carbobenzoxy -L- PHEP -L -Leu -L- Ala). In both cases there is phosphoramidate ( a covalent bond between a nitrogen and a phosphorus atom).
Technical Applications
The enzyme can be used for the synthesis of aspartame, an artificial sweetener.