Amino acid substitutions of the P2 residue of human antithrombin that either enhance or impair function.
Recombinant forms of human antithrombin (AT) were expressed in COS-1 cells, and their interaction with human thrombin characterized by comparing the reactivity of two engineered mutant forms of AT with the wild-type recombinant. Both mutant forms contained single amino acid substitutions of Asp (G392D) or Pro (G392P) for the wild-type Gly, at residue 392, termed the P2 position with reference to the adjacent reactive centre bond. All three forms of AT co-migrated on Western blots, with an apparent molecular weight of 58 kD, with endoglycosidase F treatment reducing their mobility to 47 kD. The two mutant forms of AT reacted with thrombin differently from the wild-type molecule, in that the G392D substitution abrogated the thrombin inhibitory capacity of the protein, while the G392P substitution enhanced the reactivity of the recombinant mutant AT with thrombin. Under pseudo-first order conditions, the second order rate constants for the reaction of the recombinant wild-type and G392P mutant AT were determined to be 1.4 x 10(4) L-mol-1 sec-1 and 3.0 x 10(4) L-mol-1 sec-1, respectively, a difference of 210%. In contrast, in the presence of heparin, the reaction rates of the G392P and wild-type AT forms with thrombin, differed by less than 25%. We conclude that the P2 position of AT is an important residue for AT to express its inhibitory activity, alterations to which can either enhance or impair the inhibition of thrombin by AT.
Sheffield
W P
WP
Canadian Red Cross Society Blood Services, Hamilton, Ontario.
Blajchman
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D003062:Codon / Q000235:genetics; D005347:Fibroblasts; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
D000991:Antithrombins; D003062:Codon; D011993:Recombinant Fusion Proteins; D013917:Thrombin