Redesigning the monovalent cation specificity of an enzyme.
Monovalent-cation-activated enzymes are abundantly represented in plants and in the animal world. Most of these enzymes are specifically activated by K+, whereas a few of them show preferential activation by Na+. The monovalent cation specificity of these enzymes remains elusive in molecular terms and has not been reengineered by site-directed mutagenesis. Here we demonstrate that thrombin, a Na+-activated allosteric enzyme involved in vertebrate blood clotting, can be converted into a K+-specific enzyme by redesigning a loop that shapes the entrance to the cation-binding site. The conversion, however, does not result into a K+-activated enzyme.
Prasad
Swati
S
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO 63110, USA.
Wright
Kelly J
KJ
Banerjee Roy
Dolly
D
Bush
Leslie A
LA
Cantwell
Angelene M
AM
Di Cera
Enrico
E
D001665:Binding Sites; D002414:Cations, Monovalent; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002414:Cations, Monovalent; D012964:Sodium; D013917:Thrombin; D011188:Potassium