Rational engineering of activity and specificity in a serine protease.
The discovery of the Na(+)-dependent allosteric regulation in serine proteases makes it possible to control catalytic activity and specificity in this class of enzymes in a way never considered before. We demonstrate that rational site-directed mutagenesis of residues controlling Na+ binding can profoundly after the properties of a serine protease. By suppressing Na+ binding to thrombin, we shift the balance between procoagulant and anticoagulant activities of the enzyme. Those mutants, compared to wild-type, have reduced specificity toward fibrinogen, but enhanced or slightly reduced specificity toward protein C. Because this engineering strategy targets a fundamental regulatory mechanism, it is amenable of extension to other enzymes of biological and pharmacological importance.
Dang
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Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Guinto
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di Cera
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D000595:Amino Acid Sequence; D000925:Anticoagulants; D001665:Binding Sites; D003029:Coagulants; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism
D000925:Anticoagulants; D003029:Coagulants; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D005340:Fibrinogen; D012964:Sodium; D013917:Thrombin