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Kinetics and Mechanistic Study of Hydrolysis of Adenosine Monophosphate Disodium Salt

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Kinetics and Mechanistic Study of Hydrolysis of Adenosine Monophosphate Disodium Salt Open Chem., 2019; 17: 544–556 Research Article Yoke-Leng Sim*, Beljit Kaur Kinetics and Mechanistic Study of Hydrolysis of Adenosine Monophosphate Disodium Salt (AMPNa2) in Acidic and Alkaline Media https://doi.org/10.1515/chem-2019-0044 received April 23, 2018; accepted October 22, 2018. 1 Introduction Abstract: Phosphate ester hydrolysis is essential in signal Phosphate esters are the most common chemical functional transduction, energy storage and production, information group in our body as it involves many processes in the storage and DNA repair. In this investigation, hydrolysis human body. Among some of the important processes of adenosine monophosphate disodium salt (AMPNa2) are the production of cellular energy which involves was carried out in acidic, neutral and alkaline conditions ATP and phosphenol pyruvate, essential part of nucleic of pH ranging between 0.30-12.71 at 60°C. The reaction acids, as an important component of cell membrane, and was monitored spectrophotometrically. The rate ranged most importantly storage of genetic information [1]. It is between (1.20 ± 0.10) × 10-7 s-1 to (4.44 ± 0.05) × 10-6 s-1 at often very hard for scientists to study the mechanism of [NaOH] from 0.0008 M to 1.00M recorded a second-order phosphate esters such as monoesters, diesters or triesters, -6 -1 -1 base-catalyzed rate constant, kOH as 4.32 × 10 M s . In as the cleavage rates are extremely slow in neutral acidic conditions, the rate ranged between (1.32 ± 0.06) × conditions and also due to its complicated mechanism [1]. 10-7 s-1 to (1.67 ± 0.10) × 10-6 s-1 at [HCl] from 0.01 M to 1.00 These phosphate esters are highly stable as they have an 9 M. Second-order acid-catalyzed rate constant, kH obtained estimated half-life of 3 × 10 years at pH 6.8 and 25°C and was 1.62 × 10-6 M-1 s-1. Rate of reaction for neutral region, only selected nucleases and phosphatases can accelerate -7 -1 16 21 k0 was obtained from graphical method to be 10 s . the cleavage rate by factors up to 10 and 10 [2]. Mechanisms were proposed to involve P-O bond cleavage To understand their cleavage activity, non-natural in basic medium while competition between P-O bond substrates have often been employed as mimics and N-glycosidic cleavage was observed in acidic medium. of phosphate ester linkages [2]. Among other non- In conclusion, this study has provided comprehensive natural mimics of phosphate ester linkages include bis information on the kinetic parameters and mechanism (4-nitrophenyl) phosphate (BNPP), 2-hydroxypropyl- of cleavage of AMPNa2 which mimicked natural AMP 4-nitrophenyl phosphate and so on [2,3]. In this study, cleavage and the action of enzymes that facilitate its adenosine monophosphate disodium salt (AMPNa2) was cleavage. chosen as a model substrate to mimic the phosphate ester bond in phosphate monoesters. There is extensive Keywords: Phosphate ester hydrolysis; kinetics; acid research in the recent years on adenosine and its base catalysis; P-O cleavage. corresponding nucleotides as they are biomolecules that are involved in energy production and substrates for various cellular biochemical processes [4]. Adenosine Monophosphate (AMP) is also known as 5’-adenylic acid. AMP is a nucleotide and performs the role of a monomer in RNA. The structures of AMP and adenosine are shown in Figure 1 and Figure 2 respectively. Adenosine monophosphate is present in the *Corresponding author: Yoke-Leng Sim, Universiti Tunku Abdul human body and it is broken down to adenosine by Rahman, Jalan Universiti, Bandar Barat, 31900, Kampar, Perak, Alkaline Phosphatase and ecto-5’-AMPases and endo-5’- Malaysia, E-mail: [email protected] AMPases. Natural adenosine monophosphate can also Beljit Kaur: Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar be hydrolysed into adenine and ribose 5-phosphate by Barat, 31900, Kampar, Perak, Malaysia Open Access. © 2019 Yoke-Leng Sim, Beljit Kaur, published by De Gruyter. This work is licensed under the Creative Commons Attribution alone 4.0 License. Kinetics and Mechanistic Study of Hydrolysis of Adenosine Monophosphate Disodium Salt (AMPNa2)... 545 NH 2 In light of this, it was decided to investigate the N N hydrolysis of adenosine monophosphate disodium salt (AMPNa2) (Figure 3) in alkaline and acidic conditions to O N N study the relationship between pH and the bond cleavage HO P O O reaction of the adenosine monophosphate disodium salt. OH H H This study is expected to provide insights on the effect of H H OH OH pH on the kinetics and the mechanism of the phosphate monoester bond cleavage. Figure 1: Structure of adenosine monophosphate(AMP). NH2 2 Materials and Methods N N N N 2.1 Part 1: Chemicals HO O Adenosine 5’-monophosphate disodium salt of ≥ 99.0% H H H H was purchased from Sigma Aldrich. NaCl and NaOH of OH OH the highest available purity were obtained from QRëc. TRIS and glycine of highest available purity were obtained Figure 2: Structure of adenosine. from Fisher Scientific and R&M Chemicals, respectively. NaCl served to control the ionic strength of the solutions. NH2 NaOH, HCl, TRIS, and glycine were used to vary the pH of the solutions. Infrared spectrum of AMPNa2 was obtained N N to ensure the purity of AMPNa2 before the investigation as well as to identify the structure of hydrolytic product O N N during the kinetic studies. NaO P O O ONa H H H H OH OH 2.2 Part 2: Kinetic Study of Nucleotide Analogue, AMPNa2 Figure 3: Structure of AMPNa2 . 0.01M AMPNa2 was prepared in distilled water for the kinetic studies. Sample solutions of 20 mL each consisting adenosine monophosphate nucleosidase [5]. Carrying of NaCl (to maintain ionic strength) under different buffer out studies on the adenosine monophosphate disodium conditions were prepared and thermostated at 60°C prior salt provides useful knowledge on how this enzyme to kinetic runs. A small amount of AMPNa2 (0.0001M) was carries out its function in terms of mechanism. Kinetic injected into the reaction mixture and it was immediately studies on the adenosine monophosphate disodium salt measured by UV-Vis Spectrophotometer. Subsequent using acid-base catalysis also allows the confirmation of samplings were taken until the reaction went into the principles used by these enzymes in breaking down completion of more than eight half-lives. The pH value adenosine monophosphate and therefore allowing the of each sample solution was taken before and after the development of synthetic enzymes in the future to mimic reaction completion with an EL20-Mettler Toledo pH meter. the actions of these enzymes. The enzymatic cleavage In the kinetic runs in alkaline region, the ionic strength of phosphate esters always employs general acid-base was maintained at 0.2M for [NaOH] less than 0.2M, while catalysis or metal-ion assisted catalysis where protons are for [NaOH] more than 0.2M, ionic strength was increased accepted and donated by enzyme functional groups [2,6]. to 1.0M. The rate of reaction was calculated according to General acid-base catalysis stabilizes unfavorable changes Equation 1 or Equation 2 depending on the increase or that develop in the structure during the transition state, decrease in absorbance against time for all the sample activates weak nucleophiles and stabilizes poor leaving solutions. In Equation 1 or Equation 2, Eapp is apparent groups [6]. molar extinction coefficient of the reaction mixture, A∞ is absorbance at reaction time, t = ∞, A0 is absorbance at 546 Yoke-Leng Sim, Beljit Kaur reaction time, t = 0, kobs is pseudo-first-order rate constant and [X0] represents the initial concentration of substrate, AMPNa2. Product characterization was carried out by using Fourier Transform Infrared Spectroscopy (Perkin Elmer Spectrum ex1) and Liquid Chromatography Mass Spectrometry. Aobs = Eapp [X0] exp (– kobs t) + A∞ (1) Aobs = Eapp [X0] {1-exp (– kobst)} + A0 (2) Ethical approval: The conducted research is not related to either human or animal use. Figure 4: UV-Vis absorption spectra of alkaline hydrolysis of AMPNa2 3 Results and Discussion at [NaOH] 1M at 60°C. 3.1 Part 1: Hydrolysis of AMPNa2 in Alkaline Conditions at 60°C The hydrolysis of AMPNa2 was carried out in alkaline solutions ranging from pH 9.95-12.71 at 60°C. The reaction was monitored spectrophotometrically at different time intervals until the reaction proceeded for more than 8 half- lives. Figure 4 shows typical absorption spectra of alkaline hydrolysis of AMPNa2 at [NaOH] 1.0 M and 60°C. AMPNa2 in basic solution displayed a maximum absorption peak at 260 nm which is responsible for the absorption of adenosine [7]. A reduction in the absorbance value was Figure 5: Alkaline hydrolysis of AMPNa2 in the presence of [NaOH] observed along the reaction indicating the decrease in the 1.0 M at 60°C. The solid line was drawn through the calculated data concentration of the reactant with time (Figure 5). -6 -1 points using Equation (1) with kobs = 4.44 × 10 s , Eapp = 8466 ± 44 -1 -1 The alkaline hydrolysis of AMPNa2 is following a M cm , and A∞ = 0.294 ± 0.003. pseudo-first-order kinetic model. Absorbance change with time was observed and the three parameters: kobs,Eapp and A∞ were calculated using Equation 1 for all the sample solutions covering pH 9.95-12.71. Rate constants of alkaline hydrolysis of AMPNa2 in 60°C were determined where the rate ranged from (1.20 ± 0.10) × 10-7 s-1 to (4.44 ± 0.05) × 10-6 s-1 at [NaOH] from 0.0008 M to 1.0000 M. A table consisting of [NaOH], pH, and all the kinetic parameters (kobs, Eapp, 2 A∞ and ∑di ) of this investigation is provided in SI-1 of Supporting Information.
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