Residue Asp-189 controls both substrate binding and the monovalent cation specificity of thrombin.
Residue Asp-189 plays an important dual role in thrombin: it defines the primary specificity for Arg side chains and participates indirectly in the coordination of Na(+). The former role is shared by other proteases with trypsin-like specificity, whereas the latter is unique to Na(+)-activated proteases in blood coagulation and the complement system. Replacement of Asp-189 with Ala, Asn, Glu, and Ser drastically reduces the specificity toward substrates carrying Arg or Lys at P1, whereas it has little or no effect toward the hydrolysis of substrates carrying Phe at P1. These findings confirm the important role of Asp-189 in substrate recognition by trypsin-like proteases. The substitutions also affect significantly and unexpectedly the monovalent cation specificity of the enzyme. The Ala and Asn mutations abrogate monovalent cation binding, whereas the Ser and Glu mutations change the monovalent cation preference from Na(+) to the smaller cation Li(+) or to the larger cation Rb(+), respectively. The observation that a single amino acid substitution can alter the monovalent cation specificity of thrombin from Na(+) (Asp-189) to Li(+) (Ser-189) or Rb(+) (Glu-189) is unprecedented in the realm of monovalent cation-activated enzymes.
Prasad
Swati
S
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Cantwell
Angelene M
AM
Bush
Leslie A
LA
Shih
Peter
P
Xu
Hong
H
Di Cera
Enrico
E
D000409:Alanine / Q000737:chemistry; D000495:Allosteric Site; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002412:Cations; D004305:Dose-Response Relationship, Drug; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008094:Lithium / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012413:Rubidium / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
D002412:Cations; D007477:Ions; D005973:Glutamine; D001224:Aspartic Acid; D012694:Serine; D001216:Asparagine; D008094:Lithium; D012964:Sodium; D014357:Trypsin; D013917:Thrombin; D012413:Rubidium; D000409:Alanine