Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign

Abstract

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 occurring esterases. The design was based on a previously developed strategy [G. Höst, L.G. Mårtensson, B.H. Jonsson, Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, Biochim. Biophys. Acta 1764 (2006) 1601–1606.], 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 k cat / K M = 625 (± 38) M −1 s −1. It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with k cat / K M = 101,700 (± 4800) M −1 s −1, which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable K M values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV), but for V121A/V143A/T200A no K M could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, k cat / K M is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.