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

Biochemistry
Amy E SchmidtDavid Gailani

Abstract

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 am...Continue Reading

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Citations

Jul 10, 2010·Biochimie·Peter GoettigHans Brandstetter
Aug 4, 2015·International Journal of Biological Macromolecules·Joanna Kolodziejczyk-CzepasPawel Nowak
Dec 23, 2016·Journal of Industrial Microbiology & Biotechnology·Jingjing ZhangZhiming Rao
Jun 9, 2018·Biological Chemistry·Oliver SchillingHannu Koistinen
Sep 10, 2016·Biological Chemistry·Mekdes DebelaPeter Goettig
Jul 26, 2017·Scientific Reports·R Pravin Kumar, Naveen Kulkarni

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