The reaction of chymotrypsin with its substrate was found to take place in two stages, an initial "burst" phase at the beginning of the reaction and a steady-state phase following Michaelis-Menten kinetics. The mode of action of chymotrypsin explains this as hydrolysis takes place in two steps. First, acylation of the substrate to form an acyl-enzyme intermediate, and then deacylation to return the enzyme to its original state. This occurs via the concerted action of the three-amino-acid residues in the catalytic triad. Aspartate hydrogen bonds to the N-δ hydrogen of histidine, increasing the pKa of its ε nitrogen, thus making it able to deprotonate serine. This deprotonation allows the serine side chain to act as a nucleophile and bind to the electron-deficient carbonyl carbon of the protein main chain. Ionization of the carbonyl oxygen is stabilized by formation of two hydrogen bonds to adjacent main chain N-hydrogens. This occurs in the oxyanion hole. This forms a tetrahedral adduct and breakage of the peptide bond. An acyl-enzyme intermediate, bound to the serine, is formed, and the newly formed amino terminus of the cleaved protein can dissociate. In the second reaction step, a water molecule is activated by the basic histidine, and acts as a nucleophile. The oxygen of water attacks the carbonyl carbon of the serine-bound acyl group, resulting in formation of a second tetrahedral adduct, regeneration of the serine -OH group, and release of a proton, as well as the protein fragment with the newly formed carboxyl terminus