THEORETICAL INVESTIGATION OF THE DYNAMICS OF THE ACTIVE-SITE LID IN RHIZOMUCOR-MIEHEI LIPASE

Interfacial activation of Rhizomucor miehei lipase is accompanied by a hinge-type motion of a single helix (residues 83-94) that acts as a l id over the active site. Activation of the enzyme involves the displac ement of the lid to expose the active site, suggesting that the dynami cs of the lid could be of mechanistic and kinetic importance. To inves tigate possible activation pathways and to elucidate the effect of a h ydrophobic environment (as would be provided by a lipid membrane) on t he lid opening, we have applied molecular dynamics and Brownian dynami cs techniques. Our results indicate that the lipase activation is enha nced in a hydrophobic environment. In nonpolar low-dielectric surround ings, the lid opens in approximately 100 ns in the BD simulations. In polar high-dielectric (aqueous) surroundings, the lid does not always open up in simulations of up to 900 ns duration, but it does exhibit s ome gating motion, suggesting that the enzyme molecule may exist in a partially active form before the catalytic reaction. The activation is controlled by the charged residues ARG86 and ASP91. In the inactive c onformation, ASP91 experiences repulsive forces and pushes the lid tow ard the open conformation. Upon activation ARG86 approaches ASP61, and in the active conformation, these residues form a salt bridge that st abilizes the open conformation.