IONIZABLE P-1 RESIDUES IN SERINE PROTEINASE-INHIBITORS UNDERGO LARGE PK SHIFTS ON COMPLEX-FORMATION

The burial of charged residues in proteins is rare as it is thermodynamically strongly disfavored. However, in ''standard mechanism'' protein inhibitors of serine pro teinases, the P-1 residue, which is highly exposed, be comes buried in the S-1 specificity pocket of the enzyme. In many enzymes, such as Streptomyces griseus proteinase B (SGPB) the S-1 pocket is hydrophobic. We measured the pH dependence of the association equilibrium constant for the interaction of SGPB with turkey ovo mucoid third domain P-1 mutants, Glu(18) OMTKY3 and His(18) OMTKY3. In order to eliminate the effects of other ionizable groups on the enzyme and the inhibitor, we divided these pH dependences by the pH dependence of the association equilibrium constant for the Gln(18) OMTKY3 mutant. This yielded for Glu(18), pK(f) (free inhibitor) of 4.46 +/- 0.05 and pK(c) (complex) of 8.74 +/- 0.06. For His(18) the values are: pK(f) 6.63 +/- 0.08 and pK(c) 4.31 +/- 0.07. At low pH values Glu(18) variant is a relatively good inhibitor for SGPB. This may be biologically relevant.