Inhibitory properties of the P1 Tyr variant of antithrombin.
Antithrombin (AT) and protein Z-dependent protease inhibitor (ZPI) are among two physiological serpin inhibitors in plasma that are involved in the regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has narrower protease specificity, inhibiting only factors Xa (fXa) and XIa. Unlike an Arg at the P1 site of the AT reactive center loop (RCL), this residue is a Tyr in ZPI. To investigate the contribution of P1 Tyr in restricting the specificity of ZPI, we engineered an AT mutant in which the P1 Arg of the RCL was replaced with the P1 Tyr of ZPI (AT-R393Y). The reactivity of AT-R393Y with fXa and thrombin was decreased 155- and 970-fold, respectively. However, the serpin mutant inhibited chymotrypsin with an efficiency higher by >4 orders of magnitude. By contrast, chymotrypsin did not exhibit any reactivity with ZPI. The substitution of Asp-189 of fXa with the corresponding residue of chymotrypsin (Ser) did not improve the reactivity of the protease mutant with AT-R393Y; however, the fXa mutant reacted normally with ZPI. These results suggest that the contribution of P1 Tyr to restricting the protease specificity of ZPI is RCL context-dependent and that in addition to P1 Tyr, other structural features within and/or outside the ZPI RCL are involved in determining the protease specificity of the serpin. The results further suggest that thrombin is less tolerant than fXa in accommodating the nonoptimal P1 Tyr of the AT mutant in its active-site pocket.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Manithody
Chandrashekhara
C
Qureshi
Shabir H
SH
Rezaie
Alireza R
AR
D019943:Amino Acid Substitution; D000990:Antithrombin III / Q000235:genetics* / Q000494:pharmacology; D000991:Antithrombins / Q000235:genetics* / Q000494:pharmacology; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D020134:Catalytic Domain / Q000187:drug effects; D045744:Cell Line, Tumor; D002918:Chymotrypsin / Q000494:pharmacology; D015951:Factor Xa / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011994:Recombinant Proteins; D012694:Serine / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D015843:Serpins / Q000235:genetics* / Q000378:metabolism; D013917:Thrombin / Q000235:genetics* / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D000991:Antithrombins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D000515:alpha 1-Antitrypsin; D012694:Serine; D000990:Antithrombin III; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D013917:Thrombin; D015951:Factor Xa