QM/MM metadynamics study of the direct decarboxylation mechanism for orotidine-5'-monophosphate decarboxylase using two different QM regions: acceleration too small to explain rate of enzyme catalysis

Abstract

Despite decades of study, the mechanism by which orotidine-5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine monophosphate remains unresolved. A computational investigation of the direct decarboxylation mechanism has been performed using mixed quantum mechanical/molecular mechanical (QM/MM) dynamics simulations. The study was performed with the program CP2K that integrates classical dynamics and ab initio dynamics based on the Born-Oppenheimer approach. Two different QM regions were explored. The free energy barriers for direct decarboxylation of orotidine-5'-monophosphate (OMP) in solution and in the enzyme (using the larger QM region) were determined with the metadynamics method to be 40 and 33 kcal/mol, respectively. The calculated change in activation free energy (DeltaDeltaG++) on going from solution to the enzyme is therefore -7 kcal/mol, far less than the experimental change of -23 kcal/ mol (for k(cat.)/k(uncat.): Radzicka, A.; Wolfenden, R., Science 1995, 267, 90-92). These results do not support the direct decarboxylation mechanism that has been proposed for the enzyme. However, in the context of QM/MM calculations, it was found that the size of the QM region has a dramatic effect on the calculated reaction barrier.

Figure 1

(a) The decarboxylation of OMP to UMP. (b) The carbanion intermediate formed by direct decarboxylation of OMP. (c) An overview of different mechanisms that have been proposed for the reaction in the enzyme. The mechanisms include (clockwise from top) concerted O4 protonation, Michael addition of an active site nucleophile, C5 protonation, concerted C6 protonation/decarboxylation, electrostatic stabilization of transition state, electrostatic stress of the ground state, O2 protonation, Schiff base formation with an active site Lys.

Figure 2

OMP in the active site of ODCase. The dashed lines indicate hydrogen bonding interactions taken from a molecular dynamics simulation (discussed later). cw stands for crystal water. Residue numbering is taken from the yeast enzyme.

Figure 3

OMP (grey) in the active site of ODCase. The atoms comprising the QM region used in the Carloni and coworkers QM/MM study (Raugei, S.; Cascella, M.; Carloni, P. J. Am. Chem. Soc. 2004, 126, 15730–15737).

Figure 4

OMP (grey) in the active site of ODCase. The atoms comprising the large QM region used in this study.

Figure 5

The computed energies (kcal/mol) of decarboxylation of N-methyl orotate.

Figure 6

Comparison of crystal structure and CPMD structures, with BMP bound. The crystal structure 1DQX includes bound inhibitor BMP, which is highlighted in orange. The crystal structure is shown in CPK coloring,; cw stands for crystal water. The average structure for each simulation was taken from the last picosecond of a total of 4 ps. (Left) The average structure using the large QM subsystem is shown in yellow. (Right) The average structure using the small QM subsystem is shown in yellow.

Figure 7

Metadynamics simulation of the direct decarboxylation of OMP in ODCase for the large QM subsystem. (Top) The free energy profile as a function of the CV. The first point marked on the curve, 1, is the transition structure for decarboxylation. Energies are shown relative to this point. (bottom, left). The next point on the graph, 2, is after decarboxylation has occurred and a proton is being transferred from an active site lysine to OMP (bottom, middle).The final highlighted point on the graph, 3, is the stable product (bottom, right). The error associated with the metadynamics was found to be 1.1 kcal/mol for this simulation.

Figure 8

Metadynamics simulation of the direct decarboxylation of OMP in ODCase for the Carloni QM subsystem case. (Left) The free energy profile as a function of the CV. The first point marked on the curve, 1, is the transition state for decarboxylation. Energies are shown relative to this point. (Right) A snapshot of the point on the graph, 1, is near the transition state (in the simulation) for decarboxylation.

Figure 9

The interactions that were found to be significantly different in the ground state (left) and the transition state (right) for the large and small QM subsystems. The numbers in black are the heavy atom distances from the large QM simulations, and the red numbers are the heavy atom distances from the small QM simulations.

Figure 10

Metadynamics simulation of the direct decarboxylation of OMP in solution. (Left) The free energy profile as a function of CV. The first point marked on the curve, 1, is the transition state for decarboxylation. Energies are shown relative to this point. (Right) A snapshot of the point on the graph, 1, is near the transition state (in the simulation) for decarboxylation. The error associated with metadynamics was found to be 1.6 kcal/mol for this simulation.