Fundamental reaction pathway and free energy profile for hydrolysis of intracellular second messenger adenosine 3',5'-cyclic monophosphate (cAMP) catalyzed by phosphodiesterase-4

Abstract

As important drug targets for a variety of human diseases, cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes sharing a similar catalytic site. We have performed pseudobond first-principles quantum mechanical/molecular mechanical-free energy perturbation (QM/MM-FE) and QM/MM-Poisson-Boltzmann surface area (PBSA) calculations to uncover the detailed reaction mechanism for PDE4-catalyzed hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP). This is the first report on QM/MM reaction-coordinate calculations including the protein environment of any PDE-catalyzed reaction system, demonstrating a unique catalytic reaction mechanism. The QM/MM-FE and QM/MM-PBSA calculations revealed that the PDE4-catalyzed hydrolysis of cAMP consists of two reaction stages: cAMP hydrolysis (stage 1) and bridging hydroxide ion regeneration (stage 2). The stage 1 includes the binding of cAMP in the active site, nucleophilic attack of the bridging hydroxide ion on the phosphorus atom of cAMP, cleavage of O3'-P phosphoesteric bond of cAMP, protonation of the departing O3' atom, and dissociation of hydrolysis product (AMP). The stage 2 includes the binding of solvent water molecules with the metal ions in the active site and regeneration of the bridging hydroxide ion. The dissociation of the hydrolysis product is found to be rate-determining for the enzymatic reaction process. The calculated activation Gibbs free energy of ≥16.0 and reaction free energy of -11.1 kcal/mol are in good agreement with the experimentally derived activation free energy of 16.6 kcal/mol and reaction free energy of -11.5 kcal/mol, suggesting that the catalytic mechanism obtained from this study is reliable and provides a solid base for future rational drug design.

Figure 1

(A) Division of the QM-MM systems for simulating the reaction stage 1 of PDE4-catalyzed cAMP hydrolysis. Atoms in blue are treated by the QM method. Six boundary carbon atoms (C^α, colored in red) are treated with the improved pseudobond parameters. All other atoms belong to the MM subsystem. (B to D) QM/MM-optimized geometries of key states of the reaction system for step 1, the nucleophilic attack of bridging hydroxide ion on the phosphorous atom of the cAMP. The geometries were optimized at the QM/MM(B3LYP/6-31G*:AMBER) level. The key distances in the figures are in angstrom. Carbon, oxygen, nitrogen and hydrogen atom are colored in greed, red, blue and white, respectively. The backbone of the protein is rendered in orange. The QM atoms are present as balls and sticks and the surrounding residues are rendered as sticks or lines.

Figure 2

QM/MM-optimized geometries of key states of the reaction system for step 2, the cleavage of O3'−P esteric bond. The geometries were optimized at the QM/MM(B3LYP/6-31G*:AMBER) level. See caption of Figure 1 for the color codes for various types of atoms.

Figure 3

QM/MM-optimized geometries of key states of the reaction system for step 3, the protonation of departing O3' atom. The geometries were optimized at the QM/MM(B3LYP/6-31G*:AMBER) level. See caption of Figure 1 for the color codes for various types of atoms.

Figure 4

(A) Division of the QM/MM systems for simulating the reaction stage 2. (B to D) QM/MM-optimized geometries of key states of the reaction system for the bridging hydroxide ion regeneration reaction in the active site of substrate-free PDE4. The geometries were optimized at QM/MM(B3LYP/6-31G*:AMBER) level. See caption of Figure 1 for the color codes for various types of atoms.

Figure 5

Free energy profile for the cAMP hydrolysis stage of PDE4-catalyzed hydrolysis of cAMP. The relative free energies were determined by the QM/MM-FE calculations at the B3LYP/6-31+G*:AMBER level, excluding the zero-point and thermal corrections for the QM system. Values in the parentheses are the corresponding relative free energies including the zero-point and thermal corrections for the QM subsystem. Value in the bracket is the relative free energy between ES and E'P calculated by using the QM/MM-PBSA method. Binding free energies of ES and E'P complex are estimated with QM/MM(B3LYP/6-31+G*:AMBER)-PBSA method.

Figure 6

Free energy profile for the bridging hydroxide ion regeneration reaction in the substrate-free PDE4 active site. The relative free energies were determined by the QM/MM-FE calculations at the B3LYP/6-31+G*:AMBER level, excluding the zero-point and thermal corrections for the QM system. Values in the parenthesis are the corresponding relative free energies including the zero-point and thermal corrections for the QM subsystem.

Figure 7

Reaction mechanism for the complete cycle of PDE4-catalyzed hydrolysis of cAMP. For clarity, residues His164, His200, Asp201, Asp 318, Asn321, and Gln859 are hidden from view.

Figure 8

Relative Gibbs free energies of all states of the reaction system for PDE4-catalyzed hydrolysis of cAMP. Value (-11.5 kcal/mol) in the parenthesis refers to the experimental reaction free energy reported by Goldberg et al.

Scheme 1

The catalytic mechanism proposed by Huai et al. for cAMP hydrolysis in the PDE4D active site. In the proposed mechanism, the bridging hydroxide ion will attack the phosphorous atom of cAMP, followed by the opening of phosphate ring and proton transferring from the side chain of His160 to the departing O3' atom. ES represents the cAMP-PDE4D Michaelis-Menten complex. E'P represents the complex between the enzyme and hydrolysis product AMP. The role of residue Glu339 (not shown in this scheme) was not addressed. It was unclear whether or not any intermediate(s) will be generated during the reaction.

Scheme 2

Two water molecules bind with the metal ions in the active site after the hydrolysis product leaves.