Oxidation of methionine is one of the degradation pathways of proteins. Bromate as a strong oxidizing agent can oxidize methionine to methionine sulphoxide. Computational chemistry was used to investigate the mechanism of oxidation of methionine by bromate at molecular level, using semi-empirical method at Parameterized method 3 and Modified Neglet of Diatomic Overlap level. The proposed mechanism involved seven consecutive steps. The heats of reaction of the computed proposed reaction mechanism were calculated to be -78.71kJ/mol,-3.5kJ/mol at 0oC and 25oC, respectively, at Parameterized method 3 level. While the heat of reaction based on Modified Neglect of Diatomic Overlap level of computation are -631.83kJ/mol and -533.82kJ/mol at 0oC and 25oC, respectively.The stoichiometry of the reaction was found to be 2:1 bromate to methionine, and the rate limiting step, step 2 involves the reaction between HBrO3 and methionine, which leads to the formation of intermediates that reacted and disproportionate to give the products. The equilibrium and rate constants obtained support the rate determining step.
TABLE OF CONTENTS
Table of Contents
List of Tables
List of Figures Appendix
List of Abbreviations
Mechanism of Methionine Oxidation
Physical properties of bromate.
Chemical properties of bromate
Occurrence/production of bromates
Uses of bromate
Human health issues
Bromate reservoirs, pollution incidence and its effect
Introduction to computational chemistry
The Research Problem
Justification of the Study
Aims and Objectives of the Research
Potassium Bromate and its Toxicological Effects
Causes of Protein Damage
History of computational chemistry
Methods of computational chemistry
Density functional method
Semi-empirical and empirical method
Methods for solids
Fields of application
Introduction to basis set
Minimal basis sets
Split-valence basis sets
Pople basis sets
Correlation – consistent basis sets
Other Split-valence basis sets
Plane-wave basis sets
Real-space basis sets
Interpreting Molecular Wave Functions
MATERIALS AND METHODS
Details of computational procedures
Geometry optimization of reactants, activated complexes, intermediates and products using semi-empirical methods
Calculation of HOMO and LUMO of the Reacting Species
Determination of reactivity of the optimized species: mapping of HOMO and LUMO of the optimized species.
Construction of Potential Energy Surface Diagrams
Enthalpy Change, Gibb’s Free Energy Change, Entropy Change, and Heat of Reaction.
Activation Parameters, Equilibrium, and Rate Constants
Post Computation Processing
Results of Heat of Formation, and the Thermodynamic Parameters of the Species Based on MNDO and PM3 Methods.
Results of Calculation of HOMO and LUMO.
Graphs of Heat of Formation Versus Progress of Reaction
Results of the Calculation of Heat of Reaction, Enthalpy, Entropy, and Gibb’s Free Energy Change.
Results of Calculation of Activation Parameters, Equilibrium, and Rate Constants
Optimized Geometry of Reactants, Activated Complexes, Intermediates and Products.
Calculated HOMO and LUMO of the Reacting Species
Constructed Graphs of Heat of Formation Versus Progress of Reaction
Calculated Heat of Reaction, Enthalpy, Entropy, and Gibb’s Free Energy Change.
The Rate Law
The Proposed Mechanism
The Published Mechanism
Summary, Conclusion and Recommendation
List of Tables
Table 1.1Physical properties of some common bromates.
Table 2.1 Computational methods
Table 4.2a The heat of formation and the thermodynamic parameters of the species based on PM3 level of computation.
Table 4.2bThe heat of formation and the thermodynamic parameters of the species based on MNDO level of computation.
Table 4.3 HOMO and LUMO energies of the reactants
Table 4.5a:Heat of reactions at different level of computation (from overall equation of reaction).
Table 4.5b:Heat of reaction, enthalpy, entropy, and Gibb‘s free energy change based on PM3 level of computation.
Table 4.5c: Heat of reaction, enthalpy, entropy, and Gibb‘s free energy change based on MNDO level of computation..
Table 4.6a: Activation parameters equilibrium and rate constant for various steps based on PM3 level.
Table4.6b: Activation parameters equilibrium and rate constant for various steps MNDO level.
1.1 Mechanism of Methionine Oxidation The oxidation of methionine plays an important role in vivo, during biological conditions of oxidative stress, as well as for protein stability in vitro. Depending on the nature of the oxidizing species, methionine may undergo a two-electron oxidation to methionine sulfoxide or one-electron oxidation to methionine radical cations. Both reaction mechanisms derive catalytic support from neighboring groups, which stabilize electron-deficient reaction centers. In vivo, methionine sulfoxide is subject to reduction by the methionine sulfoxidereductase (Msr) system, suggesting that some methionine sulfoxide residues may only be transiently involved in the deactivation of proteins through reactive oxygen species (ROS). Other methionine sulfoxide residues may accumulate, depending on the accessibility to Msr. Moreover, methionine sulfoxide levels may increase as a result of a lower abundance of active Msr and/or the required cofactors as a consequence of pathologies and biological aging. On the other hand, methionine radical cations will enter predominantly irreversible reaction channels, which ultimately yield carbon-centered and/or peroxyl radicals. These may become starting points for chain reactions of protein oxidation. 1.2 Bromate Bromate (BrO3-) is a negatively charged polyatomic ion containing one bromine and three oxygen atoms. Typical salts of bromate are potassium bromate(KBrO3) and sodium bromate. The acid form bromic acid is only stable in water. Many of the bromate salts are possible, but potassium bromate and sodium bromate are the most common (WHO, 2005). Table1.1 below describes these and some other available forms:
1.2.1 Physical Properties of bromates The bromates of alkali and alkaline earth metals are colourless, odourless crystals which can be in regular or powder form and have negligible vapour pressure. These salts are soluble in water and dissociate in water to the metal and bromate ions (Merk,1983;CDC, 2003; Health Canada,1999). The salts are strong oxidizers. Table 1.1summarizes the properties of some common bromates. 1.2.2 Chemical properties of bromate Bromate, thermally decompose according to the equation (1.1) and (1.2), depending on the temperature and relative stability of the bromide or oxide of metal.
………………….(1.2) Bromate is less stable than chlorate and iodate thermally. The initial step in the thermal decomposition of alkali metal bromate is the rupture of Br-O bond which occurs only at high temperature (300oC) decomposition of ammonium bromate begins at much lower temperature (-5oC). Both thermodynamically and kinetically the oxidizing power of the bromate depends on the hydrogen ion concentration. Bromates undergo some redox reactions in alkaline solutions; thus reaction with hydroxylamine or hydrazine (as their sulphates) produces nitrogen. Bromate and halides react in various possible combination, iodides are oxidized quantitatively to iodine and bromides to bromine as shown in equation (1.3) and (1.4)
……………….(1.4) Chlorides produce bromine and chlorine with bromates. Iodine replaces bromine in bromates:
The oxidation of NH3 by in perchloric acid solution affords N2, N2O, OBr-, and Br2; the oxygen in N2O is derived from the solvent. Bromates form unstable mixtures with combustible or oxidizable substances.
Table 1.1: Physical properties of some common bromates.
Colourless white granules
Colourlesscrystals,granules or crystalline powder.
75 g/L at 25oC
364 g/L at 25 oC
Solubility organic solvents.
Slightly soluble in alcohols, insoluble in ethers and acetone.
Insoluble in alcohols and ether.
1.2.3Occurrence/production of bromates
Bromate is not commonly found in water, but formed as a byproduct of ozonation disinfection of drinking water and also as a contaminant introduced from treatment of water with concentrated hypochlorite(Haag and Holgne,1993;IPCS,2000; Weinberg et al., 2003; WHO,2005;Fawell and Walker,2006). The ozonation of drinking water represents an important potential pathway of bromate formation. In such condition, drinking water is the primary route
of exposure to bromate. In Netherlands, the exposure of bromate to humans to bromate in drinking water relative to other pathways of exposure has been reported as approaching 100%(Van DijkLooijaard and Van Genderen, 2000). The bromate formation is affected by bromide concentration , pH, temperature, carbonate, alkalinity, ultra violet light, disinfectant concentration, and time(mg/L-min) and transferred ozone dose(Amy and associates,2000). Formation of bromate and haloamines after exposure to sunlight had been reported earlier in sea water to which chlorinated waste water had been discharged(Macaladyetal.,1997). Bromate was recently discovered at relatively high levels two Los Angeles reservoirs used to store water already treated by chlorine(Kemslay,2008). Water officials speculated that sunlight might have interacted with residual chlorine to oxidize bromide to bromate. The bromate level in Silver Lake and Elysian reservoirs were reported as 68 and 106 ppb, respectively. High bromate levels were reported also in two other reservoirs in San Diego County. No environmental scenario in which bromate enters the ambient air in significant quantities , although if it were present in dusts it could become airborne. Bromate salts have negligibly small vapour pressure and decompose at melting point so that they will not volatilize into the atmosphere (WHO, 2004, 2006). Bromate only slightly adsorbs to soil and its properties as strong oxidizing agent most likely lead to reactions with organic matter to form the bromide (Br-) (WHO, 2004, 2006). Bromide similarly would only slightly adsorb to soil or sediment (Health Canada, 1999).
Bromate salts are also produced intentionally, for some commercial uses. Sodium bromate is produced by the introduction of bromine into solution of sodium carbonate. Potassium bromate
is produced by passing bromine into a solution of potassium hydroxide. An industrial electrolytic process is used for large scale production of potassium bromate(IARC,1986;HSDB,1991). 1.2.4Uses of bromates Both the potassium and sodium salts of bromate are currently in commercial use worldwide. Potassium bromate, the more acutely toxic of the two, is used as a flour or bread dough ―improver‖ or maturing agent (Mack, 1998; Dupuis, 1997; WHO, 2004, 2006). Calcium bromate has also been used for this purpose. The maximum allowable level of potassium bromate in flour is75 ppm (FDA, 2007a), however, as a result of a request from the FDA in 1991, most companies have omitted potassium bromate from their products on a voluntary basis (Dupuis, 1997). Potassium bromate is also used as a chemical component of neutralizer solutions in permanent wave hair care products (Mack, 1988; DeAngeloet a; 1998; Health Canada,1999). Potassium bromate is also used in certain types of beer and cheese making (DeAngeloetal., 1998; WHO, 2005). Similarly, sodium bromate is also used in making neutralizer solutions for permanent wave hair straightening products (WEEL, 2007). While the use of the potassium bromate in these hair care preparations has significantly decreased (Mack, 1998), they remain permitted as commercial ingredients, with the maximum limits to their concentration within the cosmetic product (FDA, 2006a).
Bromate appears to be rapidly absorbed from the gastrointestinal tract at least in part unchanged, following oral administration (Fujiietal., 1984), but reduced to bromide in the body tissues.Invitro studies indicate that liver and kidney tissues degrade bromate to bromide and that glutathione (GSH) or other sulphurydyl containing compound are probably involved in that
degredation (Tanaka etal., 1984). Bromate is mainly excreted in the urine partly as bromate and partly as bromide. Some bromate may also be eliminated in the faeces(Fujiietal., 1984). Acute bromate intoxication in humans is caused by accidental or suicidal ingestion of products containing either 2% potassium bromate or 10% sodium bromate. Severe gastrointestinal irritation (vomiting,pain and dirrheoa), and CNS depression (lethargy,hypotension, hypotoxicity, and loss of reflexes) are the most common acute signs. Anemia from intravascular hemolysis may also occur. These effects are usually reversible. Later sequelae (usually within several days) include marked renal injury and hearing loss. Death from renal failure may ensue if medical intervention is not successful. If support is successful, renal function generally returns after 5-10 days. Hearing loss is usually reversible. Estimated doses in these cases ranged from about 20-1000mg BrO3-/Kg.
Bromates are formed in many different ways in municipal drinking water. The most common is the reaction ofozone and bromide is shown in equation (1.6)
Electrochemical processes, such as electrolysis of brine without a membrane operating to form hypochlorite, will also produce bromate when bromide ion is present in the brine solution.
Photo activation (sunlight exposure) will encourage liquid or gaseous chlorine to generate bromate in bromide-containing water.
In laboratories, bromates can be synthesized by dissolving Br2 in a concentrated solution of potassium hydroxide (KOH). The following reactions ( equation(1.7) and (1.8) will take place via the intermediate creation of hypobromite.
1.2.5 Human health issues
Bromate in drinking water is undesirable because it is a suspected human carcinogen(IARC, 2008;Kurokawaet al., 1990)Its presence in Coca Cola’s Dasani bottled water forced a recall of that product in the UK(BBC News,2004)
Bromate usually forms when water-containing bromide is purified using ozone, a method used at filtration plants. Proposals to reduce bromate formation include switching to enclosed atmospheric tank contact systems, lowering the water pH to between 5.9 – 6.3 , and limiting the doses of ozone. (KNBC News, 2007 ).
1.2.6 Bromate reservoirs, pollution incidence and its effect
On December 14, 2007, it was announced by the Los Angeles Department of Water and Power(LADWP) that the Silver Lake and Elysian reservoirs were going to be drained due to bromate contamination. At the Silver Lake and Elysian reservoirs, a combination of bromide from well water, chlorine and sunlight formed bromate. The decontamination took four months and resulted in the discharge of over 600 million US gallons (2.3×106 m3) of contaminated water(Neemannet al., 2004).
On June 9, 2008 the LADWP began covering the surface of the 10-acre (4 ha), 58-million-US-gallon (0.22×106 m3) open Ivanhoe reservoir with black, plastic balls to block the sunlight which causes the naturally present bromide to react with the chlorine used in treatment. It will require 30 million of the 40 cent balls ($12 million) to cover the Ivanhoe and Elysian reservoirs (Vara-Orta and Francisco, 2008). Bromate is formed when ozone used to disinfect drinking water reacts with naturally-occurring bromide found in source water. Bromate formation in disinfected drinking water is influenced by factors such as bromide ion concentration, pH of the source water, the amount of ozone and the reaction time used to disinfect the water.
The U.S. Environmental Protection Agency developed a level that it considers protective of non-cancer health effects from long-term exposure, including individuals who may be more susceptible including women of child-bearing age and children. Assuming an adult drinks about two quarts of water a day at the drinking water standard of 10 micrograms per liter, their exposure is about a sixth of that level. The increased lifetime cancer risk from drinking this water everyday poses a moderate risk level of about two in ten thousand. These exposure and
risk estimates are likely to be overestimates since most people would not consume two quarts of water-containing bromate at the standard for their lifetime. The information on the toxicity of bromate comes from accidental or intentional poisonings in people and from studies on laboratory animals. Some people who ingested large amounts of bromate had gastrointestinal symptoms such as nausea, vomiting, diarrhea and abdominal pain. Some individuals who ingested high concentrations of bromate also experienced kidney effects, nervous system effects and hearing loss. However, these people were exposed to bromate levels many thousand of times the amount that would come from drinking water at its standard. Exposure to large amounts of bromate for a long period of time caused kidney effects in laboratory animals. Long-term exposure to high levels of bromate has also caused cancer in rats. Some people may be at greater risk for developing health effects from bromate exposure or have concerns for their pregnancy or nursing infant. Because bromate can cause health effects in kidneys, it is possible that those with pre-existing kidney conditions could be at greater risk. The information on the effects of bromate on reproductive health is limited, but does not indicate a concern at levels near drinking water. 1.2.7Methionine The formula of methionine is HO2CCH(NH2)CH2CH2SCH3, and its IUPAC nomenclature is 2-amino-4-(methylthio)butanoic acid.Methionine, an essentialamino acid, is one of the two sulfur-containing amino acids. The side chain is quitehydrophobic and methionine is usually found buried within proteins. Unlike cysteine, the sulfur of methionine is not highly nucleophilic, although it will react with some electrophilic centers. It is generally not a participant in the covalent chemistry that occurs in the active centers of enzymes.
The chemical linkage of the sulfur in methionine is a thiol ether. Compare this terminology with that of the oxygen-containing ethers. The sulfur of methionine, as with that of cysteine, is prone to oxidation. The first step, yielding methionine sulfoxide, can be reversed by standard thiol- containing reducing agents. The second step yields methionine sulfone, and is effectively irreversible. It is thought that oxidation of the sulfur in a specific methionine of the
elastaseinhibitor in human lung tissue by agents in cigarette smoke is one of the causes of smoking-induced emphysema.
Methionine as the free amino acid plays several important roles in metabolism. It can react to form S-Adenosyl-L-Methionine (SAM) which serve as a methyl donor in reaction.(http://www.biology.arizona.edu) 1.2.8Methionine oxidation Methionine oxidation is a significant form of protein damage caused by endogenous or environmental oxidizing agents (Stadtman,1992).Methionine residues may be oxidized to the sulfoxide form witht-butyl hydroperoxide (tBHP) or hydrogen peroxide (H2O2) underrelatively mild conditions (Keck,1996) or to the sulfone form withother oxidizing agents under harsher conditions (Lischwe and Sung,1977). Althoughoxidation of methionine residues has no effect on the functionof some polypeptides (Glaser and Li 1974; Keck,1996), in other proteins methionineoxidation severely inhibits biological function (Chu et al., 1993;Teh et al.,1987).Since methionine oxidation can alter protein function, it is notsurprising that cells have at least two mechanisms for dealingwith proteins containing oxidized methionine residues. First,it appears likely that oxidized proteins are preferentially degradedin vivo (Stadtman,1992). Second, an enzyme termed peptidyl methionine sulfoxidereductase (MsrA), which can reduce methionine sulfoxide residuesin proteins to methionine and thus restore protein function (Moskovitzet al;996,1995), has been identified in both prokaryotes and eukaryotes. The equation below give an example of oxidation of methionine by hydrogen peroxide. Methionine(protein)+ H2O2→ Methionine Sulfoxide(protein)+ H2O Methionine Sulfoxide(protein)+ NADPH+H+→ Methionine(protein)+ NADP++H2O
1.2.9Introduction to computational chemistry
Computational chemistry is a branch of chemistry that uses principles of computerscience to assist in solving chemical problems. It uses the results of theoreticalchemistry, incorporated into efficient computerprograms, to calculate the structures and properties of molecules and solids.
The term theoretical chemistry may be defined as the mathematical description of chemistry. Currently, there are two ways to approach theoretical chemistry problems: computational theoretical chemistry and non-computational theoretical chemistry.
Computational theoretical chemistry is primarily concerned with the numerical computation of molecular electronic structures and molecular interactions and non-computational quantum chemistry deals with the formulation of analytical expressions for the properties of molecules and their reactions. The term computational chemistry is usually used when a mathematical method is sufficiently well developed that it can be automated for implementation on a computer. Computational chemistry is the application of chemical, mathematical and computing skills to solve interesting chemical problems. The quantum and classical mechanics as well as statistical physics and thermodynamics are the foundation for most of the computational chemistry theory and computer programs. This is because they model the atoms and molecules with mathematics.
Its necessity arises from the well-known fact that apart from relatively recent results concerning the hydrogenmolecularion, the quantum n-body problem cannot be solved analytically, much less in closed form. While its results normally complement the information obtained by chemical experiments, it can, in some cases, predict hitherto unobserved chemical phenomena. It is widely used in the design of new drugs and materials.
Examples of such properties are structure (i.e. the expected positions of the constituent atoms), absolute and relative (interaction) energies, electroniccharge distributions, dipoles and higher
multipole moments, vibrational frequencies, reactivity or other spectroscopic quantities, and cross sections for collision with other particles. Computational studies can be carried out to find a starting point for a laboratory synthesis, or to assist in understanding experimental data, such as the position and source of spectroscopic peaks. Computational studies can be used to predict the possibility of so far entirely unknown molecules or to explore reaction mechanisms that are not readily studied by experimental means. Thus, computational chemistry can assist the experimental chemist or it can challenge him to find entirely new chemical objects. Several major areas may be distinguished within computational chemistry: The prediction of the molecular structure of molecules by the use of the simulation of forces, or more accurate quantum chemical methods, to find stationary points on the energy surface as the position of the nuclei is varied. Storing and searching for data on chemical entities.
Identifying correlations between chemical structuresand properties. Computational approaches to help in the efficient synthesis of compounds.
Computational approaches to design molecules that interact in specific ways with other molecules (e.g. drug design and catalysis).
The methods employed in computational chemistry cover both static and dynamic situations. In all cases, the computer time and other resources (such as memory and disk space) increase rapidly with the size of the system being studied. That system can be a single molecule, a group of molecules, or a solid. Computational chemistry methods range from highly accurate to very
approximate; highly accurate methods are typically feasible only for small systems. Ab initio methods are based entirely on theory from first principles. Other (typically less accurate) methods are called empirical or semi-empirical because they employ experimental results, often from acceptable models of atoms or related molecules, to approximate some elements of the underlying theory.
Both ab initio and semi-empirical approaches involve approximations. These range from simplified forms of the first-principles equations that are easier or faster to solve, to approximations limiting the size of the system (for example, periodicboundaryconditions), to fundamental approximations to the underlying equations that are required to achieve any solution to them at all. For example, most ab initio calculations make the Born–Oppenheimerapproximation, which greatly simplifies the underlying Schrödingerequation by freezing the nuclei in place during the calculation. In principle, abinitiomethods eventually converge to the exact solution of the underlying equations as the number of approximations is reduced. In practice, however, it is impossible to eliminate all approximations, and residual error inevitably remains. The goal of computational chemistry is to minimize this residual error while keeping the calculations tractable.
In some cases, the details of electronic structure are less important than the long-time phasespace behavior of molecules. This is the case in conformational studies of proteins and protein-ligand binding thermodynamics. Classical approximations to the potential energy surface are employed, as they are computationally less intensive than electronic calculations, to enable longer simulations of moleculardynamics. Furthermore, cheminformatics uses even more
empirical (and computationally cheaper) methods like machinelearning based on physico-chemical properties. One typical problem in cheminformatics is to predict the binding affinity of drug molecules to a given target. 1.3 The Research Problem Bromate, as a strong oxidizing agent can oxidize methionine in proteins. Oxidation of methionine damages the protein in human body and pharmaceutical products, which can lead to a lot of health defects. 1.4 Justification of the Study The detailed knowledge and understanding of the mechanism of oxidation of methionine by bromate would be useful in developing better ways of controlling protein oxidation by bromates in the body and pharmaceuticals.The study will provide a more detailed mechanism that can be used for the design of formulation that will prevent and control methionine oxidation which leads to protein damage 1.5 Research Hypothesis Null hypothesis:All the published mechanisms of oxidation of methionine by bromate that involves initial uniprotonation of bromate are correct. Alternative hypothesis: The published mechanism of oxidation of methionine by bromate needs modification.
Investigation of mechanism of oxidation of methionine by bromate can be followed through experimental or theoretical technique (computational method).The two may agree with each other to give a more detailed mechanism. If not, comparing the energies of the steps involved in each method will yield a more plausible mechanism. In computational method, a more detailed
MEHANISM OF OXIDATION OF METHIONINE BY BROMATE.
SIMULATION BY COMPUTATIONAL METHOD
COMPARISON OF THE TWO METHODS
MORE PLAUSIBLE MECHANISM
MORE DETAILED PROPOSED MECHANISM
DO NOT NEED MODIFICATION
mechanism is proposed that has reactants, transition states, intermediates, and products. This can be verified experimentally as well. This leads to the modification of earlier mechanism. 1.8Aim and Objectives of the Research Aims The aim of the research is to propose a more detailed and plausible mechanism for the oxidation of methionine by bromate using computational chemistry. Objectives
1. Optimize the geometries of all the reactants, transition states, intermediates, and products involved in the proposed states.
2. Obtain the thermodynamic parameters of all the species involved.
3. Calculate the enthalpy, entropy, and Gibbs free energy change for all the steps.
4. Plot the potential energy diagrams for PM3 and MNDO computation respectively.
5. Calculate the rate constants for the various steps.
6. Locate the rate determining step from the results.
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