Hyaluronate lyase from Spectrococcus pneumonia can degrade hyaluronic acid, which is one of the major components in the extracellular matrix. The major functions of hyaluronan are to regulate water balance and osmotic pressure and act as an ion-exchange resin. In this work, we focus on the prerequisite issue of the enzymatic reaction, i.e., the initial reactive conformer. Based on the quantum mechanical and molecular mechanical molecular dynamic simulations and free energy profiles, a near attack conformer was obtained for the degradation of hyaluronan catalyzed by the hyaluronate lyase. Along with the substrate binding, the phenylhydroxyl hydrogen atom of Tyr408 will transfer to nearby His399 via a near barrierless transition state, which results in a negatively charged Tyr408 and positively charged His399. The Tyr408, rather than the previously proposed His399, was suggested to act as the general base for the subsequent beta-elimination reaction. The His399 was suggested to have the function of neutralizing the CS-carboxyl group.