How a Fragment Draws Attention to Selectivity Discriminating Features between the Related Proteases Trypsin and Thrombin

In the S1 pocket, the serine proteases thrombin and
trypsin commonly feature Asp189 and a Ala190Ser and Glu192Gln exchange.
Nevertheless, thrombin cleaves peptide chains solely after Arg, and
trypsin after Lys and Arg. Thrombin exhibits a Na+-binding site next to Asp189, which
is missing in trypsin. The fragment benzylamine
shows direct H-bonding to Asp189 in trypsin, while in thrombin, it
forms an H-bond to Glu192. A series of fragments and expanded ligands
were studied against both enzymes and mutated variants by crystallography
and ITC. The selectivity-determining features of both S1 pockets are difficult to assign to one dominating factor. The Ala190Ser
and Glu192Gln replacements may be regarded as highly conserved as
no structural and affinity changes are observed between both proteases.
With respect to charge distribution, Glu192, together with the thrombin-specific
sodium ion, helps in creating an electrostatic gradient across the
S1 pocket. This feature is definitely absent in trypsin
but important for selectivity along with solvation-pattern differences
in the S1 pocket.