title: Thrombin Glu-39 restricts the P'3 specificity to nonacidic residues.
abstract: Residue 39 of serine proteases neighbors positions P'2 to P'4 of the substrate. When Glu-39 of thrombin is replaced with Lys, the resultant enzyme (E39K) retains similar P1, P2, and P3 specificities but has altered P'3 and/or P'4 specificities. These conclusions are based on analysis of both p-nitroanilide and synthetic peptide hydrolysis. The activity of E39K is nearly normal toward 17 p-nitroanilide substrates. In peptide substrates, an acidic residue at either the P3 or P'3 position reduces the rate of cleavage by thrombin. A single substitution of Asp with Gly in either the P3 or P'3 position of a peptide corresponding to the P7-P'5 residues of protein C increases the rate of cleavage by thrombin 2-3-fold. Replacement of both Asp residues with Gly increases the rate of cleavage 30-fold. With E39K, the inhibitory effect of Asp in P3 remains unchanged, but Asp in the P'3 site is no longer inhibitory. Significant differences in the catalytic activity of E39K are also seen with respect to protein C activation. In the absence of thrombomodulin, E39K activates protein C 2.2 times faster than thrombin. In the presence of thrombomodulin, the rate of protein C activation is similar for E39K and thrombin. The second order rate constant of inhibition by antithrombin III, where P'4 is a Glu, is slightly increased (1.4-fold). The clotting activity is reduced 2.4-fold due to a lower rate of fibrinopeptides A and B release where P'3 is Arg. These data show that the P'3 position is a determinant of thrombin specificity and suggest that thrombomodulin may function in part by alleviating the inhibitory effects that may arise from the proximity of the Asp in P'3 of protein C with Glu-39 of thrombin.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
authors.forename: B F
authors.initials: BF
authors.lastname: Le Bonniec
authors.forename: R T
authors.initials: RT
authors.lastname: MacGillivray
authors.forename: C T
authors.initials: CT
authors.lastname: Esmon
mesh_terms: D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000494:pharmacology; D001483:Base Sequence; D004252:DNA Mutational Analysis; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005971:Glutamates; D009841:Oligonucleotides; D011994:Recombinant Proteins; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D000990:Antithrombin III; D013917:Thrombin