Abstract 1805 A Computational Study of Enzymatic Starch-Hydrolysis by a Soy Bean Amylase

Starches are a necessary component in the metabolic processes of plants and are a major source of nutrition for humans. Starch is broken down by a number of enzymes including amylases, which hydrolyze the starch chains into simple sugars or short glucan chains in both humans and plants. Most species have multiple copies of amylase genes each with similar structure but with a distinct function. While there is a general proposal for the chemical mechanism of amylases, it does not explain the functional and biochemical differences. A combination of X-ray crystallographic and kinetic studies revealed a general mechanism wherein amylases use one or two glutamic acids to activate water and hydrolyze the glycoside bond between sugars. We hypothesize that subtle changes in the amylase active site alter the environment and ability of water to attack the linkage, leading to changes in the rate of product formation. We are using both classical molecular dynamics (MD) and combined quantum-mechanics/molecular -mechanics (QM/MM) methods to interrogate this hypothesis employing soybean beta-amylase 5 (BAM5) as a model system. We studied this enzyme/substrate system using molecular dynamics, with the AMBER MD software program and the Amber-ff14SB and GLYCAM06EP force fields, and using QM/MM, with Gaussian09 software and the M06-2X/6-31G(d)//ff14SB method. Of particular interest was the hydrogen bonds formed by GLU186 in BAM5, since they are proposed to play a key role in catalysis. Our models demonstrated a <20% interaction probability with the O4 of the center binding glucan ring. Preliminary results express the absence of a lytic water and a highly susceptible hydrogen bonding environment. This is an important result, since the accepted mechanism implies a stronger hydrogen bond between GLU186 and the bridging oxygen and requires a lytic water. Reconciling these differences with proposed mechanisms could allow for the creation of amylases engineered to break down a variety of starches on an industrial scale and can also lead to projects focused on reprogramming amylases in other plants to increase their survivability and other efficiency within other facets,as the structure of a model organism can commit functionality between others. Further experimentation seeks to gain a better grasp on the hydrogen bonding environment to gain deeper insight into the bioinformatics of the amylase. Beyond this, comparative computation is being pursued between BAM5 and other BAM and mutations for conservative structures is being explored as the transition state of BAM5 is being sought out.