Thrombin site-directed mutagenesis

Another serine protease inhibitor of the al-antitrypsin family (serpin) is heparin cofactor II (HCII), which also forms a 1 1 complex with thrombin, but does not react with factor Xa [4,10]. The rate of inhibition of thrombin is not only increased by heparinoids but also by the related glycosaminoglycan dermatan sulfate. The identification of an inhibitor variant and site-directed mutagenesis studies on HC II cDNA led to the understanding that the binding sites for heparin and dermatan sulfate may be overlapping but not identical. Further proteinase inhibitors interacting with heparinoids are tissue factor pathway inhibitor and protease nexin-1. 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

It is not necessarily the strength of monovalent cation binding that determines the activating ability. This was demonstrated by Di Cera and coworkers. They were able to convert thrombin from a sodium-specific enzyme to a potassium-specific enzyme. This was done by site-directed mutagenesis by redesigning a loop in the backbone that defines the geometry near the cation-binding site and controls access to it. They found, however, that, while the mutant enzyme bound K+ better than Na+, it was still preferentially activated by K+ rather than Na+. 

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