Asparagine peptide lyase are one of the seven groups in which

protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the ...

s, also termed proteolytic enzymes, peptidases, or proteinases, are classified according to their catalytic residue. The

catalytic mechanism Enzyme catalysis is the increase in the rate of a process by a biological molecule, an "enzyme". Most enzymes are proteins, and most such processes are chemical reactions. Within the enzyme, generally catalysis occurs at a localized site, calle ...

of the asparagine peptide lyases involves an

asparagine Asparagine (symbol Asn or N) is an α-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), an α-carboxylic acid group (which is in the depro ...

residue acting as nucleophile to perform a nucleophilic elimination reaction, rather than

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 ...

, to catalyse the breaking of a

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 ...

. The existence of this seventh catalytic type of proteases, in which the peptide bond cleavage occurs by self-processing instead of hydrolysis, was demonstrated with the discovery of the

crystal structure In crystallography, crystal structure is a description of the ordered arrangement of atoms, ions or molecules in a crystal, crystalline material. Ordered structures occur from the intrinsic nature of the constituent particles to form symmetric pat ...

of the self-cleaving precursor of the Tsh autotransporter from ''E. coli''.

Synthesis

General asparagine self-cleaving mechanism in asparagine peptide lyases

These enzymes are synthesized as precursors or propeptides, which cleave themselves by an autoproteolytic reaction. The self-cleaving nature of asparagine peptide lyases contradicts the general definition of an enzyme given that the enzymatic activity destroys the enzyme. However, the self-processing is the action of a proteolytic enzyme, notwithstanding the enzyme is not recoverable from the reaction.

Active site and catalytic mechanism

All the proteolytic activity of the asparagine peptide lyases is only self-cleavages, then no further peptidase activity occurs. The main residue of the

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...

is the asparagine and there are other residues involved in the

, which are different between the different families of asparagine peptide lyases. The cleavage mechanism consists in the

cyclization A cyclic compound (or ring compound) is a term for a compound in the field of chemistry in which one or more series of atoms in the compound is connected to form a ring. Rings may vary in size from three to many atoms, and include examples where al ...

of the asparagine, assisted by other active site residues. In certain conditions, the asparagine cyclic structure nucleophilically attacks its

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 ...

peptide bond to the main chain forming a new bond to create a stable

succinimide Succinimide is an organic compound with the formula (CH2)2(CO)2NH. This white solid is used in a variety of organic syntheses, as well as in some industrial silver plating processes. The compound is classified as a cyclic imide. It may be prepared ...

, cleaving itself from the main chain and consequently releasing the two halves of the product.

Inhibition

No inhibitors are known.

Classification

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 ...

protease database includes the following ten families of asparagine peptide lyases, which are included in 6 different clans of proteases. Proteolytic enzymes are classified into families based on sequence similarity. Each family includes proteolytic enzymes with homologous sequences and common catalytic type. Clans are groups of proteolytic enzymes families with related structures, where catalytic type is not conserved. *Not yet included in

IUBMB The International Union of Biochemistry and Molecular Biology (IUBMB) is an international non-governmental organisation concerned with biochemistry and molecular biology. Formed in 1955 as the International Union of Biochemistry (IUB), the union ...

recommendations.

Distribution and types

The ten different families of asparagine peptide lyases are distributed in three different types: * Viral coat proteins * Autotransporter proteins * Intein-containing proteins There are five families of viral coat proteins (N1, N2, N8, N7 and N5), two families of autotransporter proteins (N6 and N4) and three families of intein-containing proteins (N9, N10 and N11).

Viral coat proteins

There are five families of viral coat proteins in which processing occurs at an asparagine residue. These five families are included in three clans: Clan NA (Families N1, N2 and N8), clan NC (Family N7) and clan NE (Family N5). Family N1: The known autolytic cleavage is mediated by the nodavirus endopeptidase, from the C-terminus of the coat protein and only occurs within the assembled

virion A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's 1 ...

. Family N2: Includes tetraviruses endopeptidases. The known autolytic cleavage is from the C-terminus of the coat protein. The cleavage occurs during the late stages of virion assembly. Family N8: The known autolytic cleavage is in

poliovirus A poliovirus, the causative agent of polio (also known as poliomyelitis), is a serotype of the species ''Enterovirus C'', in the family of ''Picornaviridae''. There are three poliovirus serotypes: types 1, 2, and 3. Poliovirus is composed of an ...

VP0 viral capsid protein into VP2 and Vp4 in the provirion. Family N7: The known autolytic cleavage is from the

N-terminus 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 ...

of the coat protein. Family N5: The known autolytic cleavage is from the

of the coat protein.

Autotransporter proteins

Tsh-associated self-cleaving domain (Escherichia coli) and similar

Autotransporter proteins are outer membrane or secreted proteins found in a broad variety of

Gram-negative bacteria Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...

. These proteins contain three structural motifs: a signal sequence, a passenger domain located at the N-terminal, and a translocator or

autotransporter domain In molecular biology, an autotransporter domain is a structural domain found in some bacterial outer membrane proteins. The domain is always located at the C-terminal end of the protein and forms a beta-barrel structure. The barrel is oriented in ...

located at the C-terminal, forming a

beta barrel In protein structures, a beta barrel is a beta sheet composed of tandem repeats that twists and coils to form a closed toroidal structure in which the first strand is bonded to the last strand (hydrogen bond). Beta-strands in many beta-barrels are ...

structure. These structures promote the protein self-transport. Autotransporter proteins are usually related to virulence functions. This fact, their interaction with host cells and the broad occurrence of autotransporter encoding genes, bring up the possibility to represent therapeutic targets for the design of vaccines against Gram-negative pathogens. Two of the families in which the

database classifies asparagine peptide lyases are autotransporter proteins, families N4 and N6. Family N4 includes secreted virulence factors, or autotransporters, from enterobacteria. Their only proteolytic activity is releasing the virulence factor from the precursor, enabling it to be secreted. The active site residues in family N4 asparagine peptide lyases are N1100, Y1227, E1249 and R1282. Family N6 includes autoprocessing

endopeptidases Endopeptidase or endoproteinase are proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule), in contrast to exopeptidases, which break peptide bonds from end-pieces of terminal amino acids. For this re ...

involved in type III protein secretion system, in which autoproteolysis is essential for mediating the secretion of proteins. Type III secretion system secretes proteins directly into host cells by an injectisome, a hollow tubular structure that penetrates into the host cell. Secreted proteins can pass through the

injectisome The type III secretion system (T3SS or TTSS), also called the injectisome, is one of the bacterial secretion systems used by bacteria to secrete their effector proteins into the host's cells to promote virulence and colonisation. The T3SS is a ...

into the host cell cytoplasm. The conserved active site residue in family N6 asparagine peptide lyases is N263.

Intein-containing proteins

An intein is a protein contained within another protein, the extein. Parasitic DNA infects an intein gene, which encodes an

endonuclease Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...

. The resulting

cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...

(complementary DNA) encodes the extein along with the intein. The intein contains a self-cleaving domain, which has the endonuclease nested within it. The intein domain performs two proteolytic cleavages at its own

and

C-terminus 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 ...

and releases from the extein, separating it in two fragments. This two fragments are then spliced together and the extein remains as a completely functional protein. The N-terminal residue of the intein domain must be a

serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...

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� ...

cysteine Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. When present as a deprotonated catalytic residue, sometime ...

, and it attacks its preceding peptide bond in order to form an ester or a thioester. The first residue of the second portion of the extein must be a serine, threonine or cysteine as well, and this second nucleophile forms a branched intermediary. The C-terminal residue of the intein domain is always an asparagine, which cyclizes to form a succinimide, cleaving its own peptide bond and releasing the intein from the extein. Finally, in the extein the

ester In chemistry, an ester is a compound derived from an oxoacid (organic or inorganic) in which at least one hydroxyl group () is replaced by an alkoxy group (), as in the substitution reaction of a carboxylic acid and an alcohol. Glycerides ar ...

thioester In organic chemistry, thioesters are organosulfur compounds with the functional group . They are analogous to carboxylate esters () with the sulfur in the thioester playing the role of the linking oxygen in the carboxylate ester, as implied by t ...

bond is rearranged to form a normal peptide bond. There are three known families of intein-containing proteins (N9, N10 and N11) all of them included in the PD clan, which contains proteolytic enzymes of different catalytic types. The tertiary structure has been solved for the intein V type proton ATPase catalytic subunit (''Saccharomyces cerevisiae''), a member of family N9 and for several inteins from family N10.

References

External links

International Proteolysis Society

Protease cut sites graphical interface

Merops - the peptidase database
*

The Proteolysis Map The Proteolysis MAP (PMAP) is an integrated web resource focused on proteases. Rationale PMAP is to aid the protease researchers in reasoning about proteolytic networks and metabolic pathways. History and funding PMAP was originally created at ...

TopFIND protease database covering cut sites, substrates and protein termini

(see als

* {{Portal bar, Biology, border=no Enzymes