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

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• 作者：Gunnar E. Hö ; st ; Bengt-Harald Jonsson
• 关键词：Carbonic anhydrase ; Hydrolysis ; Specificity ; Mutagenesis ; Protein engineering ; Rational design
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2008
• 期刊代码：161_15709639
• 类别：bio
• 出版时间：May 2008
• 卷：1784
• 期：5
• 页码：811-815
• 文件大小：424 K

摘要

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.