Structural role of Gly(193) in serine proteases: investigations of a G555E (GLY193 in chymotrypsin) mutant of blood coagulation factor XI.

ABSTRACT

In serine proteases, Gly(193) is highly conserved with few exceptions. A patient with inherited deficiency of the coagulation serine protease factor XI (FXI) was reported to be homozygous for a Gly(555) --> Glu substitution. Gly(555) in FXI corresponds to Gly(193) in chymotrypsin, which is the numbering system used subsequently. To investigate the abnormality in FXI(G193E), we expressed and purified recombinant FXIa(G193E), activated it to FXIa(G193E), and compared its activity to wild type-activated FXI (FXIa(WT)). FXIa(G193E) activated FIX with approximately 300-fold reduced k(cat) and similar K(m), and hydrolyzed synthetic substrate with approximately 10-fold reduced K(m) and modestly reduced k(cat). Binding of antithrombin and the amyloid beta-precursor protein Kunitz domain inhibitor (APPI) to FXIa(G193E) was impaired approximately 8000- and approximately 100000-fold, respectively. FXIa(G193E) inhibition by diisopropyl fluoro-phosphate was approximately 30-fold slower and affinity for p-aminobenzamidine (S1 site probe) was 6-fold weaker than for FXIa(WT). The rate of carbamylation of NH(2)-Ile(16), which forms a salt bridge with Asp(194) in active serine proteases, was 4-fold faster for FXIa(G193E). These data indicate that the unoccupied active site of FXIa(G193E) is incompletely formed, and the amide N of Glu(193) may not point toward the oxyanion hole. Inclusion of saturating amounts of p-aminobenzamidine resulted in comparable rates of carbamylation for FXIa(WT) and FXIa(G193E), suggesting that the occupied active site has near normal conformation. Thus, binding of small synthetic substrates or inhibitors provides sufficient energy to allow the amide N of Glu(193) to point correctly toward the oxyanion hole. Homology modeling also indicates that the inability of FXIa(G193E) to bind antithrombin/APPI or activate FIX is caused, in part, by impaired accessibility of the S2' site because of a steric clash with Glu(193). Such arguments will apply to other serine proteases with substitutions of Gly(193) with a non-glycine residue.