Details

Autor(en) / Beteiligte

• Page, Michael J
• Wong, Sui-Lam
• Hewitt, Jeff
• Strynadka, Natalie C. J
• MacGillivray, Ross T. A

Titel

Engineering the Primary Substrate Specificity of Streptomyces griseus Trypsin

Ist Teil von

• Biochemistry (Easton), 2003-08, Vol.42 (30), p.9060-9066

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

2003

Quelle

MEDLINE

Beschreibungen/Notizen

• Streptomyces griseus trypsin (SGT) was chosen as a model scaffold for the development of serine proteases with enhanced substrate specificity. Recombinant SGT has been produced in a Bacillus subtilis expression system in a soluble active form and purified to homogeneity. The recombinant and native proteases have nearly identical enzymatic properties and structures. Four SGT mutants with alterations in the S1 substrate binding pocket (T190A, T190P, T190S, and T190V) were also expressed. The T190P mutant demonstrated the largest shift to a preference for Arg versus Lys in the P1 site. This was shown by a minor reduction in catalytic activity toward an Arg-containing substrate (k cat reduction of 25%). The crystal structures of the recombinant SGT and the T190P mutant in a complex with the inhibitor benzamidine were obtained at high resolution (∼1.9 Å). The increase in P1 specificity, achieved with minimal effect on the catalytic efficiency, demonstrates that the T190P mutant is an ideal candidate for the design of additional substrate specificity engineered into the S2 to S4 binding pockets.