Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains
2006 (English)In: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, ISSN 1570-9639, Vol. 1764, no 10, 1601-1606 p.Article in journal (Refereed) Published
The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is > 500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.
Place, publisher, year, edition, pages
2006. Vol. 1764, no 10, 1601-1606 p.
Carbonic anhydrase, Hydrolysis, Specificity, Mutagenesis, Protein engineering, Rational design
Engineering and Technology
IdentifiersURN: urn:nbn:se:liu:diva-12859DOI: 10.1016/j.bbapap.2006.07.010OAI: oai:DiVA.org:liu-12859DiVA: diva2:17225
Gunnar Höst, Lars-Göran Mårtensson and Bengt-Harald Jonsson, Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, 2006, Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, (1764), 10, 1601-1606.