title: Conversion of trypsin into a Na(+)-activated enzyme.
abstract: Serine proteases of the chymotrypsin family show a dichotomous amino acid distribution for residue 225. Enzymes carrying Tyr at position 225 are activated by Na(+), whereas those carrying Pro are devoid of Na(+) binding and activation. Previous studies have demonstrated that the Y225P conversion is sufficient to abrogate Na(+) activation in several enzymes. However, the reverse substitution P225Y is necessary but not sufficient to introduce Na(+) binding and activation. Here we report that Streptomyces griseus trypsin, carrying Pro-225, can be engineered into a Na(+)-activated enzyme by replacing residues in the 170, 186, and 220 loops to those of coagulation factor Xa. The findings represent the first instance of an engineered Na(+)-activated enzyme and a proof of principle that should enable the design of other proteases with enhanced catalytic activity and allosteric regulation mediated by monovalent cation binding.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, Missouri 63110, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Page
authors.forename: Mark R
authors.initials: MR
authors.lastname: Bleackley
authors.forename: Steve
authors.initials: S
authors.lastname: Wong
authors.forename: Ross T A
authors.initials: RT
authors.lastname: MacGillivray
authors.forename: Enrico
authors.initials: E
authors.lastname: Di Cera
mesh_terms: D004789:Enzyme Activation / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011392:Proline / Q000378:metabolism; D015202:Protein Engineering; D012964:Sodium / Q000502:physiology*; D013305:Streptomyces griseus / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D014443:Tyrosine; D011392:Proline; D012964:Sodium; D014357:Trypsin