Functional role of residue 193 (chymotrypsin numbering) in serine proteases: influence of side chain length and beta-branching on the catalytic activity of blood coagulation factor XIa.
In serine proteases, Gly193 (chymotrypsin numbering) is conserved with rare exception. Mutants of blood coagulation proteases have been reported with Glu, Ala, Arg or Val substitutions for Gly193. To further understand the role of Gly193 in protease activity, we replaced it with Ala or Val in coagulation factor XIa (FXIa). For comparison to the reported FXIa Glu193 mutant, we prepared FXIa with Asp (short side chain) or Lys (opposite charge) substitutions. Binding of p-aminobenzamidine (pAB) and diisopropylfluorphosphate (DFP) were impaired 1.6-36-fold and 35-478-fold, respectively, indicating distortion of, or altered accessibility to, the S1 and oxyanion-binding sites. Val or Asp substitutions caused the most impairment. Salt bridge formation between the amino terminus of the mature protease moiety at Ile16 and Asp194, essential for catalysis, was impaired 1.4-4-fold. Mutations reduced catalytic efficiency of tripeptide substrate hydrolysis 6-280-fold, with Val or Asp causing the most impairment. Further studies were directed toward macromolecular interactions with the FXIa mutants. kcat for factor IX activation was reduced 8-fold for Ala and 400-1100-fold for other mutants, while binding of the inhibitors antithrombin and amyloid beta-precursor protein Kunitz domain (APPI) was impaired 13-2300-fold and 22-27000-fold, respectively. The data indicate that beta-branching of the side chain of residue 193 is deleterious for interactions with pAB, DFP and amidolytic substrates, situations where no S2'-P2' interactions are involved. When an S2'-P2' interaction is involved (factor IX, antithrombin, APPI), beta-branching and increased side chain length are detrimental. Molecular models indicate that the mutants have impaired S2' binding sites and that beta-branching causes steric conflicts with the FXIa 140-loop, which could perturb the local tertiary structure of the protease domain. In conclusion, enzyme activity is impaired in FXIa when Gly193 is replaced by a non-Gly residue, and residues with side chains that branch at the beta-carbon have the greatest effect on catalysis and binding of substrates.
Schmidt
Amy E
AE
UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery and Molecular Biology Institute, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
Sun
Mao-fu
MF
Ogawa
Taketoshi
T
Bajaj
S Paul
SP
Gailani
David
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016564:Amyloid beta-Protein Precursor / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology; D001550:Benzamidines / Q000737:chemistry; D002219:Carbamates / Q000737:chemistry; D005164:Factor IX / Q000378:metabolism; D015945:Factor XIa / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular
D016564:Amyloid beta-Protein Precursor; D000991:Antithrombins; D001550:Benzamidines; D002219:Carbamates; D007531:Isoflurophate; C006500:4-aminobenzamidine; D005164:Factor IX; D015945:Factor XIa