Conformation and Catalysis in Lysozyme. A Computational Study

Abstract

Lysozyme is an historically significant enzyme: first discovered by Alexander Fleming in 1922, it was the first enzyme to be sequenced that contains all twenty amino acids, the first to have its 3-dimensional structure determined—via X-ray crystallography—and the first to have a specific chemical mechanism suggested for its means of catalytic action. Despite its paradigmatic status and prominence in biochemistry textbooks, there is still much controversy surrounding key features of the lysozyme system. Chiefly, this concerns the role of substrate distortion in productively binding N-acetylmuramic acid (MurNAc) at the −1 sub-site of the enzyme, and the nature of the intermediate species formed in the reaction following glycosylation. Molecular dynamics (MD) simulation techniques are well-suited to exploring the conformational dynamics of highly flexible molecules like carbohydrates, whilst hybrid quantum mechanical/molecular mechanical (QM/MM) energy methods allow simulation of the reaction mechanism in the wild-type enzyme with its natural substrate, enabling vital geometries on the potential energy surface such as the transition state to be analysed directly. In this thesis, MD simulations have been employed to perform an extensive dynamical study, with both periodic and stochastic boundary conditions, of the conformational changes that MurNAc undergoes in the active site of hen egg-white lysozyme (HEWL). The thesis shows that substrate distortion is essential to binding in lysozyme, and a skew conformation of the pyranose ring has been identified as the most favourable form for binding. Semi-empirical QM/MM modelling of the reaction mechanism in HEWL has also been performed, and predicts the spontaneous formation of a covalent bond between the enzyme and MurNAc. A barrier of approximately 15 kcal/mol has been calculated for formation of the intermediate, which is in good agreement with an experimentally determined barrier of 17.8 kcal/mol. The PM3CARB-1 semi-empirical QM method for carbohydrates has also been applied to the lysozyme system.