Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign
2008 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1784, no 5, 811-815 p.Article in journal (Refereed) Published
Enzymes capable of benzoate ester hydrolysis have several potential medical and industrial applications. A variant of human carbonic anhydrase II (HCAII) was constructed, by rational design, that is capable of hydrolysing para-nitrophenyl benzoate (pNPBenzo) with an efficiency comparable to some naturally occuring esterases. The design was based on a previously developed strategy, in which docking of a transition state analogue (TSA) to the active site of HCAII was used to predict mutations that would allow the reaction. A triple mutant, V121A/V143A/T200A, was thus constructed and shown to hydrolyze pNPBenzo with kcat/KM = 625 (± 38) M-1s-1. It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with kcat/KM = 101700 (± 4800) M-1s-1, which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable KM values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV), but for V121A/V143A/T200A no KM could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, kcat/KM is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.
Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi , 2008. Vol. 1784, no 5, 811-815 p.
Carbonic anhydrase, Hydrolysis, Mutagenesis, Protein engineering, Rational design, Specificity
IdentifiersURN: urn:nbn:se:liu:diva-11787DOI: 10.1016/j.bbapap.2008.02.007OAI: oai:DiVA.org:liu-11787DiVA: diva2:18208
Original publication: Gunnar E. Höst and Bengt-Harald Jonsson, Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign, 2008, Biochimica et Biophysica Acta, (1784), 5, 811-815. http://dx.doi.org/10.1016/j.bbapap.2008.02.007. Copyright: Elsevier B.V., http://www.elsevier.com/2008-05-132008-05-132013-09-12