Regulation of the interfacial activation within the Candida rugosa lipase family

The fungus Candida rugosa produces several lipase isoenzymes and the 3D structure of three was solved (Lip1, Lip2 and Lip3). In an aqueous solution, the hydrophobic catalytic cavity of these lipases is buried under a flap that blocks the entrance of the substrate. In the hydrolysis of triacetin, the limiting step of the catalytic process was the activation of the enzyme and only the existence of the highly hydrophobic interface provided by hexane was able to shift the equilibrium towards the open conformation. In the case of Lip1, the hexane interface was crucial and once the open conformation was stabilised, Lip1 was as efficient as Lip3 for the hydrolysis of triacetin. Lip2 isoenzyme behaves more similarly to mLip3 reinforcing the higher structural and functional similarity between these isoenzymes. Inhibition experiments carried out under non-kinetic conditions allowed to correlate the higher flexibility of the closed flap and the higher hydrophobicity of the catalytic pocket of mLip3 with the greater facility of this isoenzyme to become activated by interfaces of different chemical nature. Both factors might allow a more intense penetration of mLip3 into the interfaces. In these systems, we observed a unique behaviour of Lip2, enzyme that although in a monomeric state and provide with an analogous flap structure to that of Lip1 or Lip3 underwent a very fast activation even in the absence of supramicellar concentrations of surfactants. We determined that the inhibitor itself forms micelles and hypothesised that they might provide an adequate interface for Lip2 activation. Copyright (C) 2009 John Wiley & Sons, Ltd.