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 catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...

s, also termed proteolytic enzymes, peptidases, or proteinases, are classified according to their

catalytic residue 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, the ''binding si ...

. The

catalytic mechanism Enzyme catalysis is the increase in the rate of a process by an "enzyme", a biological molecule. 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 ordered arrangement of atoms, ions, or molecules in a crystalline material. Ordered structures occur from intrinsic nature of constituent particles to form symmetric patterns that repeat ...

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, the ''binding s ...

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 ...

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, carboxy 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 t ...

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 inhibito ...

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 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 virion (plural, ''viria'' or ''virions'') is an inert virus particle capable of invading a Cell (biology), cell. Upon entering the cell, the virion disassembles and the genetic material from the virus takes control of the cell infrastructure, t ...

. 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 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, numbered 1, 2, and 3. Poliovirus is composed ...

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 amin ...

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

of the coat protein.

Autotransporter proteins

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

Gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...

. 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 (β barrel) is a beta sheet (β 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 ...

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 ...

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) 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. While the type III secretion system has been ...

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

Intein-containing proteins

intein Protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment (called an intein) is removed from a precursor protein with a ligation of C-terminal and N-terminal external proteins (called exteins) on b ...

is a protein contained within another protein, the

extein Protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment (called an intein) is removed from a precursor protein with a ligation of C-terminus, C-terminal and N-terminus, N-terminal external protei ...

. Parasitic DNA infects an intein gene, which encodes an

endonuclease In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain (namely DNA or RNA). Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (with regard to sequence), while man ...

. The resulting

cDNA In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...

(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, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein Proteins are large biomolecules and macromolecules that comp ...

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 − ...

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 when dissolved in water), a carboxyl group (which is in the deprotonated −COO− ...

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

, 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 acid (either organic or inorganic) in which the hydrogen atom (H) of at least one acidic hydroxyl group () of that acid is replaced by an organyl group (R). These compounds contain a distin ...

thioester In organic chemistry, thioesters are organosulfur compounds with the molecular structure . They are analogous to carboxylate esters () with the sulfur in the thioester replacing oxygen in the carboxylate ester, as implied by the thio- prefix ...

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) was an integrated web resource focused on proteases. Its domain now links to a scam/spam browser extender. Rationale PMAP was designed to aid the protease researchers in reasoning about proteolytic networks and metab ...

TopFIND protease database covering cut sites, substrates and protein termini

(see als

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