Mechanism of Cdc25B Phosphatase with the Small Molecule Substrate p-Nitrophenyl Phosphate from QM/MM-MFEP Calculations

Abstract

Cdc25B is a dual-specificity phosphatase that catalyzes the dephosphorylation of the Cdk2/CycA protein complex. This enzyme is an important regulator of the human cell cycle, and has been identified as a potential anti-cancer target. In general, protein tyrosine phosphatases are thought to bind the dianionic form of the phosphate and employ general acid catalysis via the Asp residue in the highly conserved WPD-loop. However, the Cdc25 phosphatases form a special subfamily based on their distinct differences from other protein tyrosine phosphatases. Although Cdc25B contains the (H/V)CX 5 R catalytic motif present in all other protein tyrosine phosphatases, it lacks an analogous catalytic acid residue. No crystallographic data currently exists for the complex of Cdc25B with Cdk2/CycA, so in addition to its natural protein substrate, experimental and theoretical studies are often carried out with small molecule substrates. In an effort to gain understanding of the dephosphorylation mechanism of Cdc25B with a commonly used small molecule substrate, we have performed simulations of the rate-limiting step of the reaction catalyzed by Cdc25B with the substrate p-nitrophenyl phosphate using the recently developed QM/MM Minimum Free Energy Path method (Hu, et al., J. Chem. Phys. 2008, 034105). We have simulated the first step of the reaction with both the monoanionic and dianionic forms of the substrate, and our calculations favor a mechanism involving the monoanionic form. Thus, Cdc25 may employ a unique dephosphorylation mechanism among protein tyrosine phosphatases, at least in the case of the small molecule substrate p-nitrophenyl phosphate.

Conical intersections in solution: Formulation, algorithm, and implementation with combined quantum mechanics/molecular mechanics method

“…44,68 Details of the sequential optimizations and their applications can be found in recent papers. 42,44,65,66,[68][69][70] In general the region that is relevant to electronicstructure change is placed into the QM subsystem; thereby, decoupling the QM conical intersection optimizations from the MM minimizations is reasonable.…”

“…68 Its remarkably enhanced efficiency is recently demonstrated by several successful simulations of enzyme catalysis. 65,66,69,70 In our QM conical intersection optimization and MM sampling scheme, we use this sequential optimization algorithm with some modifications below:…”

“…13,26,[44][45][46][63][64][65][66][67] In order to make the QM/MM minimization in solution or macromolecules efficient, we have developed a sequential optimization method where the QM and MM optimizations are sequentially, not concurrently, carried out. 44,[64][65][66][68][69][70] In this method, expensive but accurate optimization methods, such as Newton-Raphson and rational functional optimization methods, are used for optimizations of the QM subsystem, while efficient optimization methods, such as steepest descent and conjugated gradient methods, are used for minimizations of the MM subsystem. 44 As chemical reactions mainly proceed in the QM subsystem, transition state optimizations mainly depend on degrees of freedom of the QM subsystem.…”

“…42,62,68,70,72,73 This improvement obviates the QM/MM-FE requirement of defining a minimum energy reaction path prior to free energy calculations. 65,66,68,69 Recently, conical intersections of potential energy surfaces for reactions in solution or macromolecules have received much attention. [27][28][29][74][75][76][77] However, conical intersection optimizations in solution or macromolecules is more difficult than those in gas phase due to the large dimensionality of potential energy surfaces.…”

Understanding Enzyme Catalysis Using Computer Simulation

“…Case study 2: Cdc25B Phosphatase 166 Here, the emphasis is on using experimental data from site-directed mutagenesis, kinetics measurements, pH-rate profiles, and isotope effects measurements in conjunction with a QM/MM free energy simulation to provide insight into an enzymatic reaction mechanism.…”