Enzymes: Biological Catalysts And Their Applications

Classification of Enzymes

Enzymes are biodegradable, non-toxic molecules that are abundantly produced by microorganisms and have recently been produced through industrial processes. Synthetic procedures involve Protein Engineering And Recombinant DNA technology that produce efficient enzymes. The loss of catalytic activity of the enzyme is a result of treating the molecules with extreme pH or temperature, and denaturing agents (Bhatia, 2018). Enzymes are known as biological catalysts or biocatalysts that facilitate biochemical reactions. Enzymes can be extracted from cells and it can be utilised for catalysis of various commercially important procedures (Genome.gov, 2022). They are capable of facilitating reactions even in low concentrations and their characteristic is speeding up reactions by maintaining their structure (Robinson, 2015). These biocatalysts are naturally produced in the body and aid in several processes such as destruction of toxins, building muscles and break down of food particles in the digestion process, among others. The shape of an enzyme is associated with its function. For several years it was believed by researchers that enzymes are solely proteins; it was later discovered that RNA molecules are also capable of facilitating catalytic reactions. These were termed “ribozymes” that played a crucial role in the gene expression. Biochemists were also able to develop the process that enabled them to produce antibodies possessing the property of catalysis. These molecules were termed “abzymes” that were utilised significantly in therapy and as an industrial catalyst (Robinson, 2015).

Enzymes have found their application in various domains such as agriculture, food, textiles, paper that have resulted in tremendous reduction in costs. They are being utilized extensively due to their characteristic of remaining in their active state despite being removed from the environment (Bhatia, 2018). The International Union of Biochemistry and Molecular Biology classified enzymes on the basis of the catalysed reaction. Enzymes are classified into seven groups namely- Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases and translocases (McDonald and Tipton, 2021; Conix, 2020). Oxidoreductases catalyse oxidation-reduction reactions. They involve dioxygen as electron acceptor and the oxidized substrate is considered as the hydrogen donor. The enzymes under this group have names that signal the function that will be performed by them- such as ‘dehydrogenase’ or ‘oxidase’ attached to their names. For example, the reaction of ethanol for the formation of aldehyde is performed by an oxidoreductase called alcohol dehydrogenase (Pereira et al., 2018).

Transferases catalyse the reaction of transferring a group such as a glycosyl or methyl group from a donor to an acceptor, reactions such as transamination, phosphotransferase. Example of a reaction catalysed by transferases is the transfer of thiol esters by coenzyme A transferase. Hydrolases perform catalytic reactions that involve the hydrolytic cleavage of C-O, C-C and other bonds. Hydrolases acting on glycosyl, ester, amide or other bonds catalyse transfer of groups to appropriate molecules along with the hydrolytic removal of a specific group from the substrates. For example, reactions involving peptidases, phosphatases, lipases and nucleosidases- esterases cleave lipid ester bonds and phosphatases perform the catalysis of phosphate groups from molecules. Lyases break C-C-, C-N and other bonds through elimination that leaves double rings or bonds. For example, tryptophan synthase catalyses the reaction involved in the synthesis of tryptophan. Isomerases perform reactions that involve structural or geometric changes in the molecules involved. The enzymes under this group may be racemases, epimerases, mutases and others. For example, alanine racemase facilitates the interconversion of L-alanine to the isomeric form D-alanine. Ligases catalyse the reaction of “ligation” or joining together of two molecules that is coupled with the cleavage of a diphosphate bond in ATP or a similar triphosphate using water. For example, DNA ligase facilitates the reaction of forming a covalent phosphodiester linkage that permanently joins nucleotides together. Translocases assist in the transport of molecules across cell membranes. This class of enzymes are capable of catalysing movement of molecules or ions across membranes (IUBMB, 2018).  There are several roles that enzymes play in the biological system. For example, in the digestive system, enzymes facilitate proper digestion. The three primary types of digestive enzymes that are produced by the pancreas are categorized as protease, amylase and lipases. Amylases aid in breaking down carbohydrates and starches into simpler form- sugar. Whereas proteases facilitate the break down of proteins into their primary unit- amino acids; Moreover, fats and oils or lipids are digested by lipases into fatty acids and glycerol (Janiak, 2016).

Roles of Enzymes in the Digestive System

The active site binding of an enzyme and its substrate is an interaction that is highly specific. Active sites are “pockets” on these molecules that are specific for a particular substrate. The amino acids from different polypeptide chain are joined in the tertiary structure forming the protein’s folded state that make up the active sites. The substrates bind to the active site initially, through non covalent interactions; this binding is highly specific in a way that binding of an ‘improper’ molecule will not facilitate the reaction intended for that enzyme. Multiple mechanisms accelerate the conversion to the appropriate product after the specific substrate has bound to the enzyme’s active site. The enzyme has been proposed to act as a template for the substrate binding through facilitating the proper positioning on the enzyme. The acceleration of reactions by enzymes is brought about by the alteration in the conformation of the substrates to achieve their transition state. The substrate -enzyme interaction is explained through models such as the lock and key model. This model described the interaction between substrate and the enzyme as the substrate precisely fitting on the enzyme’s active site. On the other hand, the induced fit model of this interaction describes the process as modification in the configurations of the substrate and enzyme which is brought about by the substrate binding (Cooper, 2007). This model explains the modification as an alteration that closely resembles the transition state. Moreover, the distortion leas to the acceleration of the substrate conversion to the transition state, due to weakened bonds. This specific binding stabilizes the transition state, therefore decreasing the energy of activation that is required. Enzymes also require small molecules that aid in enhancing the activity of the enzyme. These small molecules are called coenzymes as they work in collaboration with enzymes to increase the rate of the reaction. Unlike substrate, coenzymes do not get altered irreversibly by the reaction, instead they get recycled to allow their participation in different reactions involving enzymes. They also act as carriers of various chemical groups such as NAD⁺ that acts as a carrier of electrons in redox reactions (Cooper, 2007).

Several factors affect the activity of an enzyme on its substrate. Factors such as the concentration of the enzyme, concentration of the substrate, presence of inhibitors, temperature, pH, the presence of activators and the concentration of the product. The concentration of the enzyme influences the activity through proportionately increasing the velocity of the reaction (Jamil et al., 2018). The concentration of the substrate affects the enzyme activity directly as the presence of more substrates for a particular number of enzymes will increase the enzyme’s activity; until a rate that is limiting the enzyme is reached, which implies the saturation of the enzyme’s active sites with the available substrates. Beyond this point, any increase in the concentration of the substrate does not produce any significant alteration in the rate of the reaction. Therefore, the excess molecules of substrate are unable to react with the enzymes as there is no available site for its binding (Jee et al., 2018). Temperature affects the enzyme through affecting the configuration of the enzyme; as majority are protein molecules this confers a sensitivity to changes in the temperature of the reaction environment. This sensitivity to temperature changes limits the enzyme reactions to facilitate in narrow temperature range- between 37 degrees to 40 degrees Celsius. For example, rise in temperature causes the enzyme activity to decrease eventually completely halting due to the drastic change in its composition. On the other hand, a decrease in temperature below the optimal range lowers the enzyme activity but this effect can be reversed once the temperature is gradually increased (Alsawmahi et al., 2018). Similarly, changes in pH affect the enzyme activity through influencing the basic amino groups and acidic carboxylic groups. Therefore, changes in pH values affect the enzymes. pH values are specific for each enzyme which allows them to efficiently function. The enzyme activity decreases to gradually stop functioning if the pH rises or falls below the optimal pH (Li et al., 2020). Activators affect the enzyme by enhancing their activity. Enzymes require inorganic metallic cations such as Mg²⁺, Ca²⁺, Cl^– and others (Supuran, 2018). On the other hand, inhibitors negatively affect the enzyme activity by competing with the substrate or through other mechanism that affect the binding, thereby affecting the activity (Bisswanger, 2019). Product concentration affects the enzyme activity through decreasing the enzyme velocity; as they combine with the enzyme’s active site resulting in the formation of a loose complex that inhibits the enzyme from performing the activity (Tang et al., 2020).

Active Site Binding of Enzymes and Substrates

The velocity of the reaction at 15 minutes was calculated using the formula (A0-A15/15), the obtained values were tabulated as shown above.

In enzyme kinetics, V_max is the maximum velocity of the reaction at which the enzymes present in the reaction environment are saturated with the substrate. K_M is the substrate concentration at which the enzyme is enabled to reach half of maximum velocity or V_max (Silverstein, 2019). The Michaelis-Menten equation is the equation for the rate of a single-substrate enzyme catalysed reaction. The equation associates the initial rate of the reaction denoted by V₀, the maximum reaction rate or V_max and the initial concentration of the substrate through the Michaelis constant K_M (Srinivasan, 2021; Andersen et al., 2018). The equation is as below-

V₀ = V_max [S] / K_M + [S]

From the graph, it was observed that the K_M was calculated to be 25 mM.

Vo = 15, [S] = 40 mM, v, Km= 25, Vmax=  ?

We know,

According to the Michaelis-Menten equation,

V= Vmax [S]/ K_M + [S]

15 = Vmax [40]/ 25+ [40]

Or, Vmax [40] = 15 x 65

Or, Vmax= 975/40

Or, Vmax= 24.4 umoles/min

Therefore, the calculated Vmax was found to be 24.4 umoles/min and the expected Vmax was 25.8 umoles/min. As the Vmax was calculated manually, the anomaly in the calculated value was observed.  

References:

Alsawmahi, O.N., Al-Juhaimi, F.Y., Alhamdan, A.M., Ghafoor, K., Mohamed Ahmed, I.A., Hassan, B.H., Ehmed, K.A., Abdelkarim, D., Younis, M., Alashmawe, N. and Adiamo, O.Q., 2018. Enzyme activity, sugar composition, microbial growth and texture of fresh Barhi dates as affected by modified atmosphere packaging. Journal of food science and technology, 55(11), pp.4492-4504.

Andersen, M., Kari, J., Borch, K. and Westh, P., 2018. Michaelis–Menten equation for degradation of insoluble substrate. Mathematical biosciences, 296, pp.93-97.

Bhatia, S. and Bhatia, S., 2018. Introduction to enzymes and their applications. Introduction to pharmaceutical biotechnology, 2, pp.1-29.

Bisswanger, H., 2017. Enzyme kinetics: principles and methods. John Wiley & Sons.

Conix, S., 2020. Enzyme classification and the entanglement of values and epistemic standards. Studies in History and Philosophy of Science Part A, 84, pp.37-45.

Cooper, G.M., Hausman, R.E. and Hausman, R.E., 2007. The cell: a molecular approach (Vol. 4, pp. 649-656). Washington, DC, USA:: ASM press.

Genome.gov, 2022. Enzyme. [online] Genome.gov. Available at: https://www.genome.gov/genetics-glossary/Enzyme

IUBMB, 2022. Enzyme Classification. [online] Iubmb.qmul.ac.uk. Available at: https://iubmb.qmul.ac.uk/enzyme/rules.html

Jamil, N., Man, R.C., Suhaimi, S., Shaarani, S.M., Arshad, Z.I.M., Mudalip, S.K.A. and Sulaiman, S.Z., 2018. Effect of enzyme concentration and temperature on the immobilization of cyclodextrin glucanotransferase (CGTase) on hollow fiber membrane. Materials Today: Proceedings, 5(10), pp.22036-22042.

Jee, A.Y., Dutta, S., Cho, Y.K., Tlusty, T. and Granick, S., 2018. Enzyme leaps fuel antichemotaxis. Proceedings of the National Academy of Sciences, 115(1), pp.14-18.

McDonald, A.G. and Tipton, K.F., 2021. Enzyme Nomenclature and Classification: the State of the Art. The FEBS journal.

Pereira, A.R., Sedenho, G.C., Souza, J.C.D. and Crespilho, F.N., 2018. Advances in enzyme bioelectrochemistry. Anais da Academia Brasileira de Ciências, 90, pp.825-857.

Petchey, M.R. and Grogan, G., 2019. Enzyme?catalysed synthesis of secondary and tertiary amides. Advanced Synthesis & Catalysis, 361(17), pp.3895-3914.

Robinson, P.K., 2015. Enzymes: principles and biotechnological applications. Essays in biochemistry, 59, p.1.

Silverstein, T.P., 2019. When both Km and Vmax are altered, Is the enzyme inhibited or activated?. Biochemistry and Molecular Biology Education, 47(4), pp.446-449.

Srinivasan, B., 2021. A guide to the Michaelis–Menten equation: steady state and beyond. The FEBS Journal.

Supuran, C.T., 2018. Carbonic anhydrase activators. Future medicinal chemistry, 10(5), pp.561-573.

Tang, J., Zhang, L., Zhang, J., Ren, L., Zhou, Y., Zheng, Y., Luo, L., Yang, Y., Huang, H. and Chen, A., 2020. Physicochemical features, metal availability and enzyme activity in heavy metal-polluted soil remediated by biochar and compost. Science of the Total Environment, 701, p.134751.

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