Details

Autor(en) / Beteiligte

• Wells, James A.
• Powers, David B.
• Bott, Richard R.
• Graycar, Thomas P.
• Estell, David A.

Titel

Designing Substrate Specificity by Protein Engineering of Electrostatic Interactions

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 1987-03, Vol.84 (5), p.1219-1223

Ort / Verlag

Washington, DC: National Academy of Sciences of the United States of America

Erscheinungsjahr

1987

Quelle

MEDLINE

Beschreibungen/Notizen

• Protein engineering of electrostatic interactions between charged substrates and complementary charged amino acids, at two different sites in the substrate binding cleft of the protease subtilisin BPN′, increases kcat/Kmtoward complementary charged substrates (up to 1900 times) and decreases kcat/Kmtoward similarly charged substrates. From kinetic analysis of 16 mutants of subtilisin and the wild type, the average free energies for enzyme-substrate ion-pair interactions at the two different sites are calculated to be -1.8 ± 0.5 and -2.3 ± 0.6 kcal/mol (1 cal = 4.18 J) [at 25 degrees C in 0.1 M Tris· HCl (pH 8.6)]. The combined electrostatic effects are roughly additive. These studies demonstrate the feasibility for rational design of charged ligand binding sites in proteins by tailoring of electrostatic interactions.