Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign.
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.
Höst
Gunnar E
GE
Molecular Biotechnology/IFM Linköping University, SE-581 83 Linköping, Sweden.
Jonsson
Bengt-Harald
BH
D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
D004952:Esters; D050505:Mutant Proteins; D004950:Esterases; D024402:Carbonic Anhydrase II