Thrombin specificity: further evidence for the importance of the beta-insertion loop and Trp96. Implications of the hydrophobic interaction between Trp96 and Pro60B Pro60C for the activity of thrombin.
A number of thrombin mutants have been constructed to investigate the role of Trp96 and the beta-insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the beta-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr(60A)Pro(60B)Pro(60C)Trp(60D) Asp(60E)Lys(60F) of the beta-insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp96 --> Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen approximately 3 times more slowly than thrombin, with the two beta-insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting approximately 3000- and 1300-fold more slowly, respectively. The specificity constant kcat/Km for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 microM(-1) s(-1), respectively, compared to 10 and 2.5 microM(-1) s(-1) for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant Km increased approximately 6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant kcat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the beta-insertion loop is important for thrombin activity. But the mutation of Trp96 --> Ser can compensate somewhat for the loss of binding at the beta-insertion loop. The deletion of the hydrophobic interaction between Trp96 and Pro(60B)Pro(60C) appears to decrease the stability of the beta-insertion loop, thereby causing a decrease in binding efficiency.
DiBella
E E
EE
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Scheraga
H A
HA
D005340:Fibrinogen / Q000502:physiology; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; D005340:Fibrinogen; D014357:Trypsin; D013917:Thrombin