Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity

Journal Article · Tue Mar 07 00:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00431a017· OSTI ID:5516731

Henriksen, R A; Mann, K G ^[1]

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1. Univ. of Vermont, Burlington (USA)

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 N{sup 2}-(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. 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 ({sup 3}H)diisopropyl fluorophosphate stoichiometrically and hydrolyzes the elastase substrate succinyl-Ala-Ala-Pro-Leu-p-nitroanilide with a relative k{sub cat}/K{sub M} of 0.14 when compared to thrombin. This results from a 3-fold increase in K{sub M} and a 2.5-fold decrease in k{sub cat} 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.

• OSTI ID: 5516731
• Journal Information: Biochemistry; (USA), Vol. 28:5; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BLOOD COAGULATION FACTORS
BIOLOGICAL MODELS
CONGENITAL DISEASES
PATHOGENESIS
THROMBIN
MOLECULAR STRUCTURE
BIOCHEMICAL REACTION KINETICS
ENZYMATIC HYDROLYSIS
GLYCINE
MUTATIONS
THREONINE
TRITIUM COMPOUNDS
VALINE
AMINO ACIDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
COAGULANTS
DECOMPOSITION
DISEASES
DRUGS
ENZYMES
HEMATOLOGIC AGENTS
HEMOSTATICS
HYDROGEN COMPOUNDS
HYDROLASES
HYDROLYSIS
HYDROXY ACIDS
KINETICS
LYSIS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PROTEINS
REACTION KINETICS
SERINE PROTEINASES
SOLVOLYSIS
550201* - Biochemistry- Tracer Techniques