Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity.
Thrombin Quick II is one of two dysfunctional forms of thrombin derived from the previously described congenital dysprothrombin prothrombin Quick. Thrombin Quick II does not clot fibrinogen, hydrolyze p-nitroanilide substrates of thrombin, or bind N2-[5-(dimethylamino)naphthalene-1-sulfonyl]arginine N,N-(3-ethyl-1,5-pentanediyl)amide, a high-affinity competitive inhibitor of thrombin. To determine the structural alteration in thrombin Quick II, the reduced, carboxymethylated protein was hydrolyzed by a lysyl endopeptidase. A peptide not present in a parallel thrombin hydrolysate was identified by reverse-phase chromatography. The peptide was purified by rechromatography and subjected to Edman degradation which showed that Gly-558 of human prothrombin had been replaced by Val. This corresponds to a point mutation of the Gly codon GGC to GUC. This Gly residue, which is highly conserved in the chymotrypsin family of serine proteases, forms part of the substrate binding pocket for bulky aromatic and basic side chains in chymotrypsin and trypsin, respectively. However, in porcine elastase 1, the corresponding residue is threonine. Consistent with the identified structural alteration, thrombin Quick II incorporates [3H]diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative kcat/KM of 0.14 when compared to thrombin. This results from a 3-fold increase in KM and a 2.5-fold decrease in kcat for thrombin Quick II when compared to thrombin acting on the same substrate. These results and those of other investigators studying mutant trypsins support the conclusion that the catalytic activity of serine proteases is very sensitive to structural alterations in the primary substrate binding pocket.
Henriksen
R A
RA
Department of Biochemistry, University of Vermont, Burlington 05405.
Mann
K G
KG
D000595:Amino Acid Sequence; D001665:Binding Sites; D001796:Blood Protein Disorders / Q000378:metabolism; D002384:Catalysis; D003198:Computer Simulation; D005998:Glycine / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007531:Isoflurophate; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D011487:Protein Conformation; D013379:Substrate Specificity; D013917:Thrombin / Q000302:isolation & purification / Q000378:metabolism*; D014633:Valine / Q000378:metabolism*
D007531:Isoflurophate; D013917:Thrombin; C027648:thrombin Quick; D014633:Valine; D005998:Glycine