Mechanistic insights of adenine promoted activity of low-molecule tyrosine phosphatase: An ONIOM study

Highlights

• The energy barrier of the rate-limiting step reduced from 16.0 kcal/mol to 14.3 kcal/mol.

• The angle of nucleophile reaction is optimized from 141.3° to 166.9°.

• Investigation of the small regulator in LWM-PTPase by the ONIOM(QM/MM) method.

• The phosphate transfer reaction of LTP1 with and without adenine was investigated.

Abstract

The mechanistic role of adenine, which assists the catalytic activity of low-molecular weight phosphatases, has been investigated using ONIOM calculations. Results confirm the dephosphorylation step being the rate-limiting step. In the absence and presence of adenine, the energy barriers of the rate-limiting step are 16.0 kcal/mol and 14.3 kcal/mol respectively, in agreement with experimental data. The formation of the favorable hydrogen bond contributes to the decreasing of barrier energy, resulting in the optimized attacking angle of the nucleophile. These results support the notion that adenine disfavors the formation of adverse hydrogen bond, which enables more effective hydrolyzation of phosphoenzyme intermediate.

Introduction

Tyrosine phosphatases (PTPases) are a family of enzymes essential for many cellular processes, including growth, migration, proliferation, differentiation and energy metabolism [1], [2], [3], [4], [5]. The Low-molecular weight protein tyrosine phosphatases (LMW-PTPases) are single-domain cytosolic member of PTPases with molecular masses of approximately 18 kDa, existing ubiquitously in prokaryotic and eukaryotic organisms [6], [7], [8], [9]. The LMW-PTPases have the conserved substrate phosphoryl binding motif C(X)5R(S/T) known as the PTP signature motif or P-loop in the active site which also occur in two types of PTPase in protein sequence, namely high molecular weight PTPases and dual specificity phosphatases [10], [11], [12]. Hence, they share identical catalytic mechanism [13]. The LMW-PTPases can increase the expression of mRNA and protein levels in breast, colon, bladder and kidney cancer cells, which are associated with cancer progression [14], [15]. As regulators of a substantial number of signaling pathways, the LMW-PTPases were also implicated in the development of type II diabetes and obesity [16], [17], [18]. Therefore, significant therapeutic interest arises in inquiring potential factor affecting the activity of the LMW-PTPase to control its anomalous expression in humans and to provide guidance on the development of corresponding drug design [19], [20], [21], [22].

Earlier studies found that certain purines and their derivatives were able to promote catalytic efficiency of LMW-PTPases [23], [24], [25]. For instance, hydrolysis of p-Nitrophenyl Phosphate (pNPP) in the yeast LMW-PTPase was found to be enhanced by 30-fold in the presence of adenine [26]. Kinetic measurements also reveal that adenine is capable of increasing the activity of bacterial and human LMW-PTPase [8], [26], [27]. Adenine derivatives play an important role in antiviral and cytostatic activities [28]. Several effective adenine derivatives have been marketed for the treatment of HIV, HBV, CMV and other virus-infected diseases. Undoubtedly, adenine derivatives are one of the most valuable inhibitors for the development of antiviral agents. Being a biological component, adenine also provides significant inspiration and ideas for the development of stable and green bionic small molecule regulators for disease control and drug design in the future. The LMW-PTPase catalyzed phosphoryl transfer reaction follows a two-step mechanism [13]. The nucleophilic cysteine residue attacks the phosphoryl group of the substrate and leads to formation of the covalent phosphoenzyme intermediate [29], [30], followed by hydrolyzation and release of one molecule of inorganic phosphate. Hydrolyzation of the covalently bonded phosphoenzyme intermediate was generally considered as the rate-limiting step [31], [32]. Theoretically studies on the bovine protein tyrosine phosphatase (BPTP) by Bashford et al. [33] suggested that the first step of the phosphoryl transfer may follow a dissociative path with an energy barrier of 9 kcal/mol and the associative one with 22 kcal/mol. For the hydrolyzation step, the calculated energy barrier was 17.6 kcal/mol [34]. While there are experimental and simulation studies on the phosphoryl transfer reaction for the LMW-PTPases, however, the role of adenine in the phosphoryl transfer process at the molecular level remains elusive [33], [34], [35]. To the best of our knowledge, the role of adenine in phosphoryl transfer process has not been thoroughly studied by and theoretical study yet. Since quantum mechanical (QM) calculation are very computational demanding and conventional molecular mechanics (MM) doesn’t allow to simulate chemical reactions. Therefore, we explored the catalytic role of adenine for the LMW-PTPases using a state-of-the-art quantum mechanics and molecular mechanics (QM/MM) hybrid method with the two-layer calculations by using a density functional theory (DFT) B3LYP/6-31G (d, p) [36], [37], [38] and AMBER [39] theories. In this approach, DFT and MM are used together to describe the reaction mechanism within an enzyme that consists of thousands of atoms.