Conversion of trypsin into a Na(+)-activated enzyme.
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.
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Michael J
MJ
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, Missouri 63110, USA.
Bleackley
Mark R
MR
Wong
Steve
S
MacGillivray
Ross T A
RT
Di Cera
Enrico
E
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
D014443:Tyrosine; D011392:Proline; D012964:Sodium; D014357:Trypsin