QM/MM computational studies and theoretical development: The reaction mechanism of 4-oxalocrotonate tautomerase

We have studied the mechanism for the reaction catalyzed by the 4-oxalocrotonate tautomerase (4OT) enzyme as well as compared the turnover rate for two substrates, using newly developed quantum mechanical/molecular mechanical QM/MM methods. Total free energy barriers were obtained for the two steps involved in this reaction. In the first step, Pro-1 abstracts a proton from the substrate: 2-oxo-4-hexenedioate (2o4hex) or 2-hydroxymuconate (2HM), creating a negative charge on the oxygen at C2 of this substrate. In the second step, this hydrogen is returned to C5 of the substrate to form the product, 2-oxo-3-hexenedioate (2o3hex). Our calculations clearly show that there is no general acid in the reaction. Arg-39″ and an ordered water stabilize the negative charge by electrostatic interactions without acting as general acids in the reaction. Our theoretical results point to 2HM as the substrate which is turned-over faster by 4OT, however, both substrates should be taken into consideration. By performing theoretical calculations on the wild-type enzyme (wt-4OT) and a 4OT analogue that contains a backbone amide-to-ester mutation between Ile-7 and Leu-8, we are able to show that the protein backbone of 4OT plays a role in the catalysis of this enzyme.; Additionally, we have developed a combined parallel path optimizer procedure for the efficient determination of minimum energy reaction paths (MEPs) in enzymes with the ab-initio QM/MM approach. We have also developed a new method to determine the free energy profiles associated with these MEPs. The combined path optimizer procedure relies on two "chain-of-replica" methods. In this approach an initial path optimization is performed with a QM/MM implementation of the nudged elastic band (NEB) method. After this step, the path is refined with the parallel path optimizer method developed by Prof. H. Liu and Mr. Z. Lu, to obtain the converged path. The parallel path optimizer method is based on the path optimization procedure proposed by Ayala and Schlegel for small molecules in gas phase, where the reaction paths are represented by a discrete number of structures. For computational efficiency, the number of structures along the path is small. Generally seven structures are sufficient to obtain a converged path. (Abstract shortened by UMI.)...