Molecular Dynamics and Free Energy Simulations of Phenylacetate and CO Release from AMDase and Its G74C/C188S Mutant: A Possible Rationale for the Reduced Activity of the Latter.

Arylmalonate decarboxylase (AMDase) catalyzes the decarboxylation of α-aryl-α-methyl malonates to produce optically pure α-arylpropionates of industrial and medicinal importance. Herein, atomistic molecular dynamics simulations have been carried out to delineate the mechanism of the release of product molecules phenylacetate (PAC) and carbon dioxide (CO), from the wild-type (WT) and its G74C/C188S mutant enzymes. Both of the product molecules follow a crystallographically characterized solvent-accessible channel to come out of the protein interior. A higher free energy barrier for the release of PAC from G74C/C188S compared to that in the WT is consistent with the experimentally observed compromised efficiency of the mutant. The release of CO precedes that of PAC; free energy barriers for CO and PAC release in the WT enzyme are calculated to be ∼1-2 and ∼23 kcal/mol, respectively. Postdecarboxylation, CO moves toward a hydrophobic pocket formed by Pro 14, Leu 38, Leu 40, Leu 77, and the side chain of Tyr 48 which serves as its temporary "reservoir". CO releases following a channel mainly decorated by apolar residues, unlike in the case of oxalate decarboxylase where polar residues mediate its transport.