Engineering the pH-optimum of a triglyceride lipase: from predictions based on electrostatic computations to experimental results

The optimisation of enzymes for particular purposes or conditions remains a n important target in virtually all protein engineering endeavours. Here, w e present a successful strategy for altering the pH-optimum of the triglyce ride lipase cutinase from Fusarium solani pisi. The computed electrostatic pH-dependent potentials in the active site environment art: correlated with the experimentally observed enzymatic activities. At pH-optimum a distinct negative potential is present in all the lipases and esterases that we stu died so far. This has prompted us to propose the 'The Electrostatic Catapul t Model' as a model for product release after cleavage of the ester bond. T he origin of the negative potential is associated with the titration status of specific residues in the vicinity of the active site cleft. In the case of cutinase, the role of Glu44 was systematically investigated by mutation s into Ala and Lys. Also, the neighbouring Thr45 was mutated into Proline, with the aim of shifting the spatial location of Glu44. All the charge muta nts displayed altered titration behaviour of active site electrostatic pote ntials. Typically, the substitution of the residue Glu44 pushes the onset o f the active site negative potential towards more alkaline conditions. We, therefore, predicted more alkaline pH optima, and this was indeed the exper imentally observed. Finally, it was found that the pH-dependent computed Co ulombic energy displayed a strong correlation with the observed melting tem peratures of native cutinase. (C) 2001 Elsevier Science B.V. All rights res erved.