Enteropeptidase (1EKB) from Bos taurus
Created by: Davis Moore
Enteropeptidase (PDB ID: 1EKB), also known as enterokinase, is a membrane-bound serine protease found in most mammals (1). In Bos taurus, enteropeptidase exists as a two-chain polypeptide originating from a single-chain precursor, proenteropeptidase (2). The N-terminal 120 kDa heavy chain, which contains the transmembrane segment responsible for anchoring the protein, is attached by a disulfide linkage to the C-terminal 47 kDa light chain (2). The light chain of enteropeptidase carries the catalytic serine protease function and is therefore the main functional domain (2).The light chain is divided into three subunits, and the serine protease function is found in subunit B (2). Enteropeptidase can function without the heavy chain, but its function is decreased about 500-fold (2, 3).
Enteropeptidase is part of an
enzymatic cascade crucial for proper intestinal digestion in mammals (1, 2).
Enteropeptidase is produced in the duodenum and remains there bound to
enterocytes by its heavy chain (2). During digestion, pancreatic secretions
pass through the duodenum, where enteropeptidase recognizes the activation
peptide of trypsinogen (1TGB), another key digestive enzyme (2). Upon binding
to enteropeptidase, trypsinogen is cleaved to yield trypsin (1K1M), which
catalyzes the activation of most other digestive proteins, including
inactivated trypsinogen (4).
The catalytic center of
enteropeptidase is made up of the same structural elements as most other serine
proteases (2, 5). Three residues form the main catalytic site, also known as
the “catalytic triad”: Asp-102, His-57 and Ser-195 (2). This catalytic domain
interacts with the bovine trypsinogen N-terminal activation sequence Val-Asp-Asp-Asp-Asp-Lys-Ile
(4). For ease of reference, the residues of the Val-Asp-Asp-Asp-Asp-Lys are
numbered by positions P1-P6, with the valine residue being P1. Upon contact, the C-terminal trypsinogen
lysine residue (P1) becomes covalently bound to the His-57 and Ser-195 residues
of enteropeptidase: Ser-195 attacks the carbonyl carbon of Lys-P5 to form a
tetrahedral hemiketal intermediate as the methylene carbon atom of Lys-P5 binds
to the imidazole ring of His-57 (2, 5). Once bound, the enteropeptidase residue
Asp-189 binds to and neutralizes the terminal amino group of the trypsinogen Lys-P5 residue (Image 1) (2). While bound to Asp-189, Lys-P5 also participates in hydrogen
bonding with two water molecules that are commonly incorporated into the
structure of enteropeptidase and several other serine proteases (2, 5). These
hydrogen bonds are significant as they result in the aliphatic section of the Lys-P5 side-chain to move close to the main-chain sections of Phe-215 and Ser-214,
which help stabilize the tetrahedral intermediate by charge dispersion (2).
Cleavage of the trypsinogen
activation site is carried out not only by the catalytic center, but also by an
extended substrate binding exosite found on the surface of the enteropeptidase
molecule (2). The active residue in this binding exosite is Lys-99 (2). The
three aspartic acid residues directly following lysine in the trypsinogen
activation site (P2, P3, P4) surround the basic side-chain of the Lys-99
residue: Asp-P2 and Asp-P4 both form salt bridges with Lys-99, while Asp-P3
becomes hydrogen-bonded to Tyr-174 of the enteropeptidase molecule (2). At this
point, water attacks the lysine residue at P5, resulting in another tetrahedral
intermediate (2, 5). This intermediate cannot be sustained, and the bond
between Lys-P5 and Ile-P6 is broken, yielding a cleaved pentapeptide and a
molecule of activated trypsin (2, 5).
The secondary structure of
enteropeptidase was evaluated using the UniProt software provided by the ExPASy
database (6). The secondary structure of enteropeptidase is organized mainly
into beta-sheets (2, 6). The light-chain of enteropeptidase contains 13 beta-sheets,
3 alpha-helices, and one 3/10-helix (2, 6). The 3/10 –helix connects
helix α-1 and strand β-4 (2).
The tertiary structure of the enteropeptidase light-chain consists of
beta-barrels made up of six beta-sheets each (2, 6). The two barrels serve to
surround the catalytic protease domain and support the substrate binding
exosite on the surface of the molecule (2). The catalytic serine protease
domain is supported by five disulfide bonds: Cys-1-Cys-122, Cys-42-Cys-58, Cys-136-Cys-201,
Cys-168-Cys-182, and Cys-191-Cys-220 (2). The isoelectric point of
enteropeptidase is 5.13 and the molecular weight is 28348 Da (6).
The function of enteropeptidase is
not dependent on any ligand or cofactor to execute its function of trypsinogen
cleavage (2). The only ligand associated with the protein is Zn2+,
which is simply used in the X-ray crystallography structure determination (2).
The zinc ion was incorporated into the molecule between various acidic
side-chains on the outside of the molecule (2).
Enteropeptidase, as a crucial enzyme
in the intestinal digestive pathway of all mammals, has constantly been studied
since its discovery in the early 20th century (7). Though the light chain
discussed here is exclusive to Bos taurus,
it has a highly-conserved analog in all mammalian species (1). Because it is
intimately involved in the digestion of food, deficiency of enteropeptidase can
cause improper intestinal absorption in any mammalian species, especially
humans (2, 7). Additionally, enteropeptidase deficiency can cause diarrhea,
vomiting, and growth failure (2). Congenital enteropeptidase deficiency is
generally caused by mutations in the proenteropeptidase gene, which codes for
the monomeric precursor of enteropeptidase (7). Not only does enteropeptidase
catalyze the cleavage of trypsinogen into trypsin, but trypsin also catalyzes
the cleavage of proenteropeptidase into enteropeptidase (7). Therefore, an
enteropeptidase deficiency can compound itself and requires regular pancreatic
extract treatments for normal digestive function (2).
Due to its remarkable specificity,
enteropeptidase is often used in biochemical experiments to efficiently cleave
various fusion proteins (8). Enteropeptidase can be purified from the duodena
of adult cattle and used in the laboratory to cleave proteins at arbitrary
peptide sequences with the scheme Lys-Asp-Asp-Asp-Asp (8).
Matriptase (1EAX) is a human serine
protease that is expressed in human cancer and cancer-derived cells (9).
Matriptase is similar in structure to enteropeptidase, but it has a
very different role in metabolism: matriptase has been associated with the
degradation of cellular matrices and metastasis (9). In order to compare the primary
structures of matriptase and enteropeptidase, a Position-Specific-Iterated
Basic Local Alignment Search Tool (PSI-BLAST) was used (10). PSI-BLAST
identifies molecules that share sequence homology with a query protein by evaluating
differences in the primary structure of the proteins (10). The program
identifies “gaps,” which are amino acid sequences that exist in the subject
protein but not the query protein (10). The program then assigns an E value: a
low E value suggests a high level of homology, with an E value of less than .05
being significant for proteins (10). Given enteropeptidase as the query
protein, matriptase was given an E value of 2e-132, suggesting a high degree of
homology (10). Only 2% of the primary sequences of enteropeptidase and matriptase
are different (10). Matriptase has a catalytic center that is identical to that
of enteropeptidase: Asp-102, His-57 and Ser-195 form a catalytic triad that
binds its substrate (9). Matriptase also has a tertiary structure that is
similar to that of enteropeptidase, as demonstrated by a search using the Dali
server (11). The Dali server identifies proteins with a tertiary structure
similar to that of a query protein by comparing intramolecular distances within
the molecules (11). The server produces a Z score for each matched molecule,
which represents structural similarities between the query molecule and various
subject molecules (11). A Z score above 2 indicates a significant similarity;
matriptase was assigned a Z score of 40.6 when compared with enteropeptidase,
indicating a high level of structural homology (11). Because enteropeptidase
and matriptase are both functional serine proteases, their primary, secondary,
and tertiary structures are highly conserved (9, 10, 11). Enteropeptidase and
matriptase operate by almost identical mechanisms; the difference lies in the
peptide sequences that they recognize (2, 9).