In an early debate in 1969, Dahlquist proposed a covalent mechanism for lysozyme based on kinetic isotope effect, but for a long time the ionic mechanism was more accepted. In 2001, a revised mechanism was proposed by Vocadlo via a covalent but not ionic intermediate. Evidence from ESI-MS analysis indicated a covalent intermediate. A 2-fluoro substituted substrate was used to lower the reaction rate and accumulate an intermediate for characterization. The amino acid side-chains glutamic acid 35 (Glu35) and aspartate 52 (Asp52) have been found to be critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, whereas Asp52 acts as a nucleophile to generate a glycosyl enzyme intermediate. The Glu35 reacts with water to form hydroxyl ion, a stronger nucleophile than water, which then attacks the glycosyl enzyme intermediate, to give the product of hydrolysis and leaving the enzyme unchanged. This covalent mechanism was named after Koshland, who first proposed this type of mechanism.