Contributions of basic amino acids in the autolysis loop of factor XIa to serpin specificity.
The autolysis loops (amino acids 143-154, chymotrypsinogen numbering) of plasma serine proteases play key roles in determining the specificity of protease inhibition by plasma serpins. We studied the importance of four basic residues (Arg-144, Lys-145, Arg-147, and Lys-149) in the autolysis loop of the coagulation protease factor XIa (fXIa) for inhibition by serpins. Recombinant fXIa mutants, in which these residues were replaced individually or in combination with alanine, were prepared. The proteases were compared to wild-type fXIa (fXIa-WT) with respect to their ability to activate factor IX in a plasma clotting assay, to hydrolyze the chromogenic substrate S2366, and to undergo inhibition by the C1-inhibitor (C1-INH), protein Z dependent protease inhibitor (ZPI), antithrombin (AT), and alpha(1)-protease inhibitor (alpha(1)-PI). All mutants exhibited normal activity in plasma and hydrolyzed S2366 with catalytic efficiencies similar to that of fXIa-WT. Inhibition of mutants by C1-INH was increased to varying degrees relative to that of fXIa-WT, with the mutant containing alanine replacements for all four basic residues (fXIa-144-149A) exhibiting an approximately 15-fold higher rate of inhibition. In contrast, the inhibition by ZPI was impaired 2-3-fold for single amino acid substitutions, and fXIa-144-149A was essentially resistant to inhibition by ZPI. Alanine substitution for Arg-147 impaired inhibition by AT approximately 7-fold; however, other substitutions did not affect it or slightly enhanced inhibition. Arg-147 was also required for inhibition by alpha(1)-PI. Cumulatively, the results demonstrate that basic amino acids in the autolysis loop of fXIa are important determinants of serpin specificity.
Rezaie
Alireza R
AR
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA. rezaiear@slu.edu
Sun
Mao-fu
MF
Gailani
David
D
D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000737:chemistry* / Q000235:genetics; D000991:Antithrombins / Q000494:pharmacology; D001329:Autolysis / Q000235:genetics*; D050718:Complement C1 Inhibitor Protein / Q000494:pharmacology; D015945:Factor XIa / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D015843:Serpins / Q000494:pharmacology*
D024361:Amino Acids, Basic; D000991:Antithrombins; D050718:Complement C1 Inhibitor Protein; D011994:Recombinant Proteins; C416370:SERPINA10 protein, human; D015843:Serpins; D015945:Factor XIa