Biomaterial Database

Kinetic mechanism for the interaction of Hirulog with thrombin. 1994

M A Parry, and J M Maraganore, and S R Stone

Department of Haematology, University of Cambridge, U.K.

Hirulog (D-FPRPGGGGDGDFEEIPEEYL) is a bivalent inhibitor of thrombin consisting of a moiety (D-FPRP) that binds to the active-site cleft and a hirudin-like C-terminal region (DGDFEEIPEEYL) that binds to the positively charged surface groove of thrombin known as the anion-binding exosite. The formation of the thrombin-Hirulog complex was studied using steady-state and rapid kinetics at 37 degrees C. The inhibition constant for Hirulog was found to be 1.9 nM. Hirulog was slowly degraded by thrombin with a kcat value of 0.01 s-1. The formation of the complex resulted in an enhancement of 44% in the intrinsic fluorescence of thrombin. The kinetics of the increase in thrombin fluorescence were described by a double-exponential decay. The dependence of the rate constant for the fast phase on the concentration of Hirulog could be described by the Michaelis-Menten equation with Km and kmax values of 0.75 +/- 0.12 microM and 325 +/- 17 s-1. The data were consistent with a mechanism in which the C-terminal region of Hirulog binds to the anion-binding exosite with a dissociation constant of 0.75 microM in the first step, followed by two intramolecular steps with rate constants of about 300 and 30 s-1. A C-terminal fragment of hirudin was found to compete in the first step confirming that this process corresponded to the binding of the hirudin-like C-terminus of Hirulog to the anion-binding exosite.(ABSTRACT TRUNCATED AT 250 WORDS)

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008956	Models, Chemical	Theoretical representations that simulate the behavior or activity of chemical processes or phenomena; includes the use of mathematical equations, computers, and other electronic equipment.	Chemical Models,Chemical Model,Model, Chemical
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010446	Peptide Fragments	Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.	Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D005470	Fluorometry	An analytical method for detecting and measuring FLUORESCENCE in compounds or targets such as cells, proteins, or nucleotides, or targets previously labeled with FLUORESCENCE AGENTS.	Fluorimetry,Fluorometric Analysis,Analysis, Fluorometric
D006629	Hirudins	Single-chain polypeptides of about 65 amino acids (7 kDa) from LEECHES that have a neutral hydrophobic N terminus, an acidic hydrophilic C terminus, and a compact, hydrophobic core region. Recombinant hirudins lack tyr-63 sulfation and are referred to as 'desulfato-hirudins'. They form a stable non-covalent complex with ALPHA-THROMBIN, thereby abolishing its ability to cleave FIBRINOGEN.	Hirudin
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein