Biology Reference
In-Depth Information
The extensive positive electrostatic charge associated with the heparin binding
site in extant thrombin was much smaller in size and field strength in the model
of the putative ancient protein (
Figure 10.2
). In prothrombin, the second kringle
domain interacts with the heparin-binding site to slow down antithrombin
III/heparin-mediated inhibition (Arni
et al
., 1993) prior to proteolytic prothrom-
bin activation. The primitive heparin binding site evident in the ancient protease
would have been unable to bind the kringle 2 domain as strongly as its extant
descendant. This is consistent with the view that the ancient protein contained a
light chain of Gla and EGF domains, the latter only being replaced by kringles at
a later stage in the evolution of the protease (Patthy, 1985). The heparin-binding
site may then have evolved in such a way as to balance the dual requirements of
antithrombin III/heparin-mediated inhibition of thrombin and the kringle 2-
mediated protection of prothrombin from premature inhibition.
10.2.5 The evolution of human protein C
One of the best characterized vitamin K-dependent serine proteases of coagula-
tion is protein C. Activated protein C (APC) exerts a negative feedback regulatory
effect on both the intrinsic and extrinsic pathways of coagulation through the
proteolytic inactivation of factors Va and VIIIa in the presence of protein S and a
negatively charged phospholipid surface (Tuddenham and Cooper, 1994). Protein
C is activated by thrombin through cleavage of the Arg169-Leu170 bond with
release of a dodecapeptide from the heavy chain. This reaction is enhanced some
20 000-fold by thrombomodulin, an endothelial cell surface glycoprotein which
binds thrombin with high affinity (reviewed by Esmon, 1995).
The substrate specificity of serine proteases is largely dependent upon the
structure and properties of the substrate-binding pocket adjacent to the active
site. By analogy with other trypsin-like serine proteases, Asp189 (chymotrypsin
numbering) of APC forms the bottom of this binding pocket (Segal
et al
., 1971;
Shekter and Berger, 1967). In principle, the presence of Ser198 at the S2 substrate-
binding site of APC would allow the binding pocket to accommodate larger
amino acid residues such as Phe or Leu (Stone and Hofsteenge, 1985). However,
this theoretical diversity appears not to be capitalised upon by the native structure
since the substrate specificity of APC is in reality confined to factors Va and VIIIa
(Esmon, 1987).
A molecular model of the catalytic domain of early mammalian protein C was
also derived and was used to examine the functional architecture of that ancient
domain and to explore its evolutionary progression from the putative common
ancestor of the vitamin K-dependent serine proteases toward extant human pro-
tein C (Wacey
et al
., 1997). This application of homology modeling to a recon-
structed amino acid sequence made it possible to trace the evolution of structural
features in a human protein and, in so doing, to make certain inferences regard-
ing the development of its functional specificity. Since its first appearance in evo-
lution as a result of a gene duplication, the catalytic domain of human protein C
has undergone 41 amino acid changes, at least some of which have presumably
served to 'fine-tune' interactions with its substrates. Thus, those sites of protein-
protein interaction already present in early mammalian protein C should contain
