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c h a p t e r 36
Biochemistry of Hemostasis
Proteinase Domain Structures
The amino acid sequences of the proteinase domains
are homologous to the pancreatic serine proteinases chy-
motrypsin and trypsin
. 2
Primary specificity, i.e., recog-
nition of the amino acid at which the peptide bond is hy-
drolyzed, is trypsin-like. All of the coagulation proteinases
cleave peptide bonds at arginyl residues; however, plasmin
shows a preference for ly syl residues. Peptide bond hydrol-
ysis occurs in the proteinase active sites according to the
mechanism established for chymotrypsin and other ser-
ine proteinases. The amino acid residues in the active site
that are responsible for the hydrolytic reaction are Asp102,
His
5 7
and Ser195, respectively. These three residues form
the charge relay system that facilitates the attack by wa-
ter on the intermediate enzyme-substrate complex and the
splitting of the peptide bond. Within the active site, His57,
acts as a general base in the hydrolytic process. Asp102,
the other residue in the charge relay with His57, acts as
an acid. The Ser residue forms an acyl enzyme (strictly
a tetrahedral intermediate) with the arginyl peptide bond
that is being cleaved. The attack of water on the acyl en-
zyme completes the hydrolysis. Specificity for basic amino
acids is conferred by Asp
1 8 9
(not shown), which interacts
electrostatically with the guanidine group of the Arg side
chain or amino group of the Lys side chain of the protein
substrate.
In our “low-resolution” picture, the proteinase domains
differ most obviously from the pancreatic proteinases in
that they contain inserted sequences. These insertions are
found primarily on the surfaces of the proteinase do-
mains and are responsible for the high specificity that the
coagulation proteinases have for their protein substrates,
all of which are cleaved at Arg (or Lys) residues. Con-
ventional secondary structure cartoons that illustrate the
differences between trypsin and thrombin are shown in
Figure 36-4.
Amino Terminal Domain Structures and
Structural Motifs
The N-terminal domains of the hemostatic proteinase pre-
cursors and proteinases are constructed of several pro-
tein structural motifs (Figure 36-5). These motifs, in vari-
ous combinations and by placement in different positions,
2
Because of the initial identification of these residues in chymotrypsin,
their numbering follows that of chymotrypsin. Actual numbers for amino
acid residues are given in the figure legends or in the figures themselves.
molecule found at the N-terminal end provides for recog-
nition of cofactor proteins and membrane lipid surfaces.
Trypsin
Throm bin
F IG U R E 3 6 -4
(Also see color figure.) Proteinase domains of serine proteinases trypsin
and thrombin. In the color version of this figure, the active site
am in o a c id
resid u es (sh o w n a s sp a ce-fillin g stru ctu res)
are Ser195 (shown in white);
His57 (shown in blue), and Asp102 (shown in red). The
sp ecificity
pocket
residue is Asp189. Both trypsin, the reference serine proteinase, and
thrombin are shown as cartoons with the secondary structure elements
marked as defined in the coordinate files for the structures from the Protein
Data Bank. The color scheme is that of R. Sayle, the creator of RasMol, the
program used to produce these ribbon diagrams. Some of the functionally
significant differences in thrombin are the residues in the helix above the
catalytic triad and two ligand binding regions, exosites I and II (not shown).
provide the building blocks of the non-proteinase-forming
regions. With limited changes in their individual amino
acid sequences, the motifs in the different proenzymes
provide sites that are responsible for the formation of the
particular activation complexes.
The four common motifs found within the amino ter-
minal regions of the proteinase precursors and proteinases
are “kringles,” epidermal growth factor (EGF)-like mo-
tifs, fibronectin motifs, and “apple” motifs (Figure 36-5).
The kringle is so named because in two dimensions, i.e.,
on paper prior to determination of its three-dimensional
structure, it has the shape of a Danish pretzel. Similarly,
apple motifs resemble drawings of apples.
Although the many components of the hemostatic sys-
tem inevitably create a high level of complexity, the func-
tional and structural similarities between and among the
different molecules permit the simplification described
above. Simplification can be obtained by representing
many of the molecules as ellipses, the two-dimensional
representations of the generally ellipsoid shapes of the
molecules in solution (see Figure 36-9 below). However,
the common motifs and similar proteinase domain struc-
tures can be presented even more simply in the form of
bar diagrams, which are usually used in the descriptions
of reactions that follow.
Cofactor Proteins
Cofactor proteins bind both the proteinase (enzyme) and
the proenzyme (substrate). They bind to lipid membranes,
