Protein-Induced Change in Ligand Protonation during Trypsin and Thrombin Binding: Hint on Differences in Selectivity Determinants of Both Proteins?

Trypsin
and thrombin, structurally similar serine proteases, recognize
different substrates; thrombin cleaves after Arg, whereas trypsin
cleaves after Lys/Arg. Both recognize basic substrate headgroups via
Asp189 at the bottom of the S1 pocket. By crystallography and isothermal
titration calorimetry (ITC), we studied a series of d-Phe/d-DiPhe-Pro-(amino)­pyridines. Identical ligand pairs show the
same binding poses. Surprisingly, one ligand binds to trypsin in protonated
state and to thrombin in unprotonated state at P1 along with differences
in the residual solvation pattern. While trypsin binding is mediated
by an ordered water molecule, in thrombin, water is scattered over
three hydration sites. Although having highly similar S1 pockets,
our results suggest different electrostatic properties of Asp189 possibly
contributing to the selectivity determinant. Thrombin binds a specific
Na+ ion next to Asp189, which is absent in trypsin. The
electrostatic properties across the S1 pocket are further attenuated
by charged Glu192 at the rim of S1 in thrombin, which is replaced
by uncharged Gln192 in trypsin.