Thermolysin (, ''Bacillus thermoproteolyticus neutral proteinase'', ''thermoase'', ''thermoase Y10'', ''TLN'') is a

thermostable In materials science and molecular biology, thermostability is the ability of a substance to resist irreversible change in its chemical or physical structure, often by resisting decomposition or polymerization, at a high relative temperature. ...

neutral

metalloproteinase A metalloproteinase, or metalloprotease, is any protease enzyme whose catalytic mechanism involves a metal. An example is ADAM12 which plays a significant role in the fusion of muscle cells during embryo development, in a process known as myogen ...

enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...

produced by the

Gram-positive In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. Gram-positive bacte ...

bacteria ''Bacillus thermoproteolyticus''. It requires one zinc ion for enzyme activity and four

calcium ions Calcium ions (Ca2+) contribute to the physiology and biochemistry of organisms' cells. They play an important role in signal transduction pathways, where they act as a second messenger, in neurotransmitter release from neurons, in contractio ...

for structural stability. Thermolysin specifically catalyzes the

hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...

peptide bond In organic chemistry, a peptide bond is an amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein cha ...

s containing hydrophobic amino acids. However thermolysin is also widely used for peptide bond formation through the reverse reaction of hydrolysis. Thermolysin is the most stable member of a family of metalloproteinases produced by various ''

Bacillus ''Bacillus'' (Latin "stick") is a genus of Gram-positive, rod-shaped bacteria, a member of the phylum ''Bacillota'', with 266 named species. The term is also used to describe the shape (rod) of other so-shaped bacteria; and the plural ''Bacilli ...

'' species. These enzymes are also termed 'neutral' proteinases or thermolysin -like proteinases (TLPs).

Synthesis

Like all bacterial

extracellular This glossary of biology terms is a list of definitions of fundamental terms and concepts used in biology, the study of life and of living organisms. It is intended as introductory material for novices; for more specific and technical definitions ...

proteases thermolysin is first synthesised by the bacterium as a pre-proenzyme. Thermolysin is synthesized as a pre-proenzyme consisting of a

signal peptide A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16-30 amino acids long) present at the N-ter ...

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 The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...

. In the

periplasm The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the ''periplasmic space'' in gram-negative bacteria. Using cryo-electron microscopy it has been found that ...

pre-prothermolysin is then processed into prothermolysin by a

signal peptidase Signal peptidases are enzymes that convert secretory and some membrane proteins to their mature or pro forms by cleaving their signal peptides from their N-termini. Signal peptidases were initially observed in endoplasmic reticulum (ER)-deri ...

. The prosequence then acts as a

molecular chaperone In molecular biology, molecular chaperones are proteins that assist the conformational folding or unfolding of large proteins or macromolecular protein complexes. There are a number of classes of molecular chaperones, all of which function to assi ...

and leads to autocleavage of the peptide bond linking pro and mature sequences. The mature protein is then secreted into the extracellular medium.

Structure

Thermolysin has a molecular weight of 34,600 Da. Its overall structure consists of two roughly spherical

domain Domain may refer to: Mathematics *Domain of a function, the set of input values for which the (total) function is defined **Domain of definition of a partial function **Natural domain of a partial function **Domain of holomorphy of a function * Do ...

s with a deep cleft running across the middle of the molecule separating the two domains. The

secondary structure Protein secondary structure is the three dimensional conformational isomerism, form of ''local segments'' of proteins. The two most common Protein structure#Secondary structure, secondary structural elements are alpha helix, alpha helices and beta ...

of each domain is quite different, the

N-terminal domain The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the ami ...

consists of mostly beta pleated sheet, while the

C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is ...

domain is mostly

alpha helical The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...

in structure. These two domains are connected by a central alpha helix, spanning amino acids 137-151. 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. The thermal stability of members of the TLP family is measured in terms of a ''T''₅₀ temperature. At this temperature incubation for 30 minutes reduces the enzymes activity by half. Thermolysin has a ''T''₅₀ value of 86.9 °C, making it the most thermo stable member of the TLP family. Studies on the contribution of

calcium Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar to ...

to thermolysin stability have shown that upon thermal inactivation a single calcium ion is released from the molecule. Preventing this calcium from originally binding to the molecule by

mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...

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. In particular a

phenylalanine Phenylalanine (symbol Phe or F) is an essential α-amino acid with the formula . It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine. This essential amino a ...

(F) at amino acid position 63 and a

proline Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the prot ...

(P) at amino acid position 69 contribute significantly to thermolysin stability. Changing these amino acids to

threonine Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), a carboxyl group (which is in the deprotonated −COO� ...

(T) and

alanine Alanine (symbol Ala or A), or α-alanine, is an α-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side c ...

(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.

Applications

*In the synthesis of

aspartame Aspartame is an artificial non-saccharide sweetener 200 times sweeter than sucrose and is commonly used as a sugar substitute in foods and beverages. It is a methyl ester of the aspartic acid/phenylalanine dipeptide with the trade names ...

, less bitter-tasting byproduct is produced when the reaction is catalyzed by thermolysin. *Determining protein stability in cell lysate using the fast parallel proteolysis (FASTpp) assay.

References

External links

* The

MEROPS MEROPS is an online database for peptidases (also known as proteases, proteinases and proteolytic enzymes) and their inhibitors. The classification scheme for peptidases was published by Rawlings & Barrett in 1993, and that for protein inhibitor ...

online database for peptidases and their inhibitors
M04.001
* {{Portal bar, Biology, border=no EC 3.4.24 Zinc proteins Proteases