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Combined Enzyme and Substrate Design: Grafting of a cooperative two-histidine catalytic motif into a protein targeted at the scissile bond in a designed ester substrate
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
2007 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 8, no 13, 1570-1576 p.Article in journal (Refereed) Published
Abstract [en]

A histidine-based, two-residue reactive site for the catalysis of hydrolysis of designed sulfonamide-containing para-nitrophenyl esters has been engineered into a scaffold protein. A matching substrate was designed to exploit the natural active site of human carbonic anhydrase II (HCAII) for well-defined binding. In this we took advantage of the high affinity between the active site zinc atom and sulfonamides. The ester substrate was designed to position the scissile bond in close proximity to the His64 residue in the scaffold protein. Three potential sites for grafting the catalytic His-His pair were identified, and the corresponding N62H/H64, F131H/V135H and L198H/P202H mutants were constructed. The most efficient variant, F131H/V135H, has a maximum kcat/KM value of approximately 14 000 M-1 s-1, with a kcat value that is increased by a factor of 3 relative to that of the wild-type HCAII, and by a factor of over 13 relative to the H64A mutant. The results show that an esterase can be designed in a stepwise way by a combination of substrate design and grafting of a designed catalytic motif into a well-defined substrate binding site.

Place, publisher, year, edition, pages
2007. Vol. 8, no 13, 1570-1576 p.
Keyword [en]
chiral resolution, enzyme catalysis, hydrolysis, mutagenesis, protein engineering
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-12012DOI: 10.1002/cbic.200600540ISI: 000249366500014PubMedID: 17665409OAI: oai:DiVA.org:uu-12012DiVA: diva2:39781
Available from: 2007-11-16 Created: 2007-11-16 Last updated: 2011-01-25Bibliographically approved

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Baltzer, Lars
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