Quantum mechanical/molecular mechanical study of three stationary points along the deacylation step of the catalytic mechanism of elastase

A large amount of experimental as well as theoretical information is available about the mechanism of serine proteases, but many questions remain unanswered. Here we study the deacylation step of the reaction mechanism of elastase. The water molecule in the acyl-enzyme active site, the binding mode of the carbonyl oxygen in the oxyanion hole, the characteristics of the tetrahedral intermediate structure, and the mobility of the imidazole ring of His-57 were studied with quantum mechanical/molecular mechanical methods. The models are based on a recent high-resolution crystal structure of the acyl-enzyme intermediate. The nucleophilic water in the active site of the acyl-enzyme has been shown to have two minima that differ by only 2 kcalmol⁻¹ in energy. The carbonyl group of the acyl-enzyme is located in the oxyanion hole and is positioned for attack by the hydrolytic water. The tetrahedral intermediate is a weakly bonded system, which is electrostatically stabilized by short hydrogen bonds to the backbone NH groups of Gly-193 and Ser-195 in the oxyanion hole. The short distance between the N^ɛ2 of His-57 and the O^γ of Ser-195 in the tetrahedral intermediate indicates a small movement of the imidazole ring towards the product in the deacylation step. The carbonyl group of the enzyme-product complex is not held strongly in the oxyanion hole, which shows that the peptide is first released from the oxyanion hole before it leaves the active site to regenerate the native state of the enzyme. Received: 11 September 2000 / Accepted: 15 September 2000 / Published online: 21 March 2001