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Introduction to Enzymology

Enzymology

Enzymology is a field of study that deals with a specific group of proteins called enzymes. These proteins accelerate specific chemical reactions in a biological system, and these reactions are essential to the growth, development, adaptation, and survival of the organism. The absence, accumulation, or dysfunctionality of an enzyme has drastic effects on the living organism, some of which are reflected as metabolic disorders.

What are Enzymes?

Enzymes are proteins that act as catalysts in living cells, thus they speed up the rate of a specific chemical reaction in the cell. They enable metabolic reactions, which are non-spontaneous chemical reactions, to take place rapidly and in a controlled manner in living cells that would otherwise take too long in the mild cellular environment.[1]

Enzymes act specifically only on their substrate or reactant. This provides living cells with a means to control when and where certain metabolic reactions should take place.

How Do They Work?

The keys to how enzymes control cellular reactions lie in their catalytic properties and the specificity of their substrate. The catalytic properties of an enzyme determine the rate at which a certain reaction should occur, while its substrate specificity dictates which starting reactant the enzyme should react with.

Enzymes determine their substrate through their structure

Like other proteins, enzymes are made up of a particular sequence of amino acids, whose intramolecular interactions give proteins their three-dimensional shape or conformation. Together with the functional group of the amino acids on the protein surface, the protein conformation determines the type of molecules that the particular protein interacts with.[2]

Along the same line, enzymes interact with their substrates or other molecules, which are typically much smaller in size, at their active site.[3] When viewed in three dimensions, the active site is typically shaped like a pocket or cleft on the enzyme, which restricts molecules that act as the substrate only to those possessing geometric complementarity.[1,3] In addition to the complementarity in the shape, the substrate must also electronically complement the amino acid residues at the enzyme’s active site. This allows for the formation of weak and non-covalent chemical bonds between the substrate and the enzyme’s active site to initiate the enzyme’s catalytic function.[1,2]

The shape and electronic complementarity form the basis of the lock-and-key model, where the enzyme is the lock and its substrate is its compatible key. The induced-fit model is a newer model that illustrates how the enzyme and substrate interact. In this model, the active site of the enzyme, similar to the lock-and-key model, is already formed, but its conformation alters upon binding to the substrate.[1,3] In both models, the binding of the substrate to the active site of the enzyme is regarded as the starting point of enzymatic catalysis.[2]     

Enzymes lower the activation energy by stabilizing the transition state

For a chemical reaction to proceed and result in a product, the molecules of the starting reactant(s) must acquire a certain amount of energy, called activation energy, for them to start transforming into the product. Once the reactant molecules have acquired the activation energy, they exist in a short-lived, high-energy transition state, which is unstable and can either be reverted to the reactant or converted to the products of the reaction.[1] Since only the reactant molecules exist at the initiation of the reaction, the transition state is converted to the product until an equilibrium is attained.[1,2]

In analogy to climbing a mountain, whether a chemical reaction can take place and at what rate will depend on the height of the mountain, i.e., the activation energy, and how fast the reactants can climb to the top of the mountain. In the presence of a catalyst, such as an enzyme, the activation energy is lowered and the height of the mountain is subsequently decreased. Thus, the transition state of a chemical reaction with enzymatic catalysis is lower in energy than that without enzymes. As a result, the transition state of the catalytic reaction is more stabilized, allowing the reactant-product equilibrium to be reached faster than the ones without the catalyst.[1-4]

In enzyme-catalyzed reactions, the enzyme binds to its substrate(s) and facilitates the reaction by stabilizing the transition state, allowing other parts of the substrate to go forward with the reaction or bringing multiple substrates in the vicinity of the others.[4] This results in the reduction of the activation energy and subsequent breakdown of the enzyme-substrate bond. When the reaction reaches the reactant-product equilibrium, the enzyme, like a catalyst, is still present and not degraded during the process.[1-4]

Some enzymes require cofactors for their functions.

Apart from the substrate-enzyme interaction, certain enzymes may require cofactors for them to function properly. Cofactors can be inorganic metal or organic molecules, the latter of which are also termed coenzymes.

Another way of classifying cofactors is based on how they function in a catalyzed reaction. Cofactors that only interact with enzymes transiently during the reaction catalysis, typically at their active sites, are called cosubstrates. Another group of cofactors is called prosthetic groups. They are cofactors that covalently bond to the enzyme regardless of the catalysis state. The enzyme without its usual prosthetic group is generally inactive and is referred to as an apoenzyme, while the active enzyme with its prosthetic group bound to it is termed holoenzyme. A well-known example of a holoenzyme and its prosthetic group is cytochromes and heme, respectively.[1,3]

Classification and Nomenclature

Most enzymes can be recognized from the suffix -ase to the chemical reactions and their corresponding substrate. As more and more enzymes are discovered and characterized, all enzymes are given official names based on their substrate(s) and the type of reaction they catalyze.

They are also designated with a unique Enzyme Commission (EC) number, consisting of four digits that follow the EC rules. The first digit of the EC number refers to the type of reaction the enzyme catalyzes, and these reactions are classified into the following groups:[3]

   1.        EC1: Oxidoreductase

Enzymes classified in this group are involved in biological redox reactions. They function in the transfer of electrons from one substrate to another. An example of enzymes in this group is the ʟ-Lactate: NAD+ oxidoreductase (EC 1.1.2.3), commonly known as lactic dehydrogenase or cytochrome b2. It’s involved in glycolysis, in which NAD+ is generated from the reduction of pyruvate to lactate, and the reverse oxidation of lactate and NAD+ to pyruvate.[5]

   2.        EC2: Transferases

Transferase enzymes are those involved in the exchange of a functional group from one molecule to another. An example of a transferase is methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13), which is also known as methionine synthase or MetH. It is involved in the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine to regenerate methionine, forming tetrahydrofolate in the process.[6]

   3.        EC3: Hydrolases

Enzymes in the hydrolase group catalyze hydrolysis reactions by adding a water molecule to their substrate. For example, acetylcholine acetylhydrolase, also known as acetylcholinesterase (EC 3.1.1.7), catalyzes the hydrolysis of acetylcholine, resulting in the split of an acetyl group from acetylcholine into acetate and choline.[3]

   4.        EC4: Lyases

Lyases refer to enzymes that catalyze the formation of double bonds by the elimination of a functional group. Pyruvate decarboxylase (EC 4.1.1.1) is a good example of lyases. It catalyzes the decarboxylation – removal of carbon dioxide – of pyruvic acid, leading to the formation of acetaldehyde, which is a part of the ethanol fermentation process.[7]

   5.        EC5: Isomerases

Isomerases are enzymes that catalyze isomerization. ᴅ-Glyceraldehyde-3-phosphate keto isomerase (EC 5.3.1.1) is an example of an isomerase enzyme that catalyzes the formation of glyceraldehyde-3-phosphate isomer, dihydroxyacetone phosphate.[3]

   6.        EC6: Ligases

Ligases are enzymes that catalyze the formation of chemical bonds, coupled with ATP cleavage. An example of a ligase enzyme is pyruvate CO2 ligase (EC 6.4.1.1), which catalyzes the formation of oxaloacetate from the condensation of pyruvate and carbon dioxide, coupled with ATP cleavage.[3]

Examples of Enzyme-Deficiency Disorders

Since enzymes are essential to the organism’s biochemical reactions, deficiencies or abnormalities in the function of an enzyme will have a direct impact on the organism’s metabolism, resulting in metabolic syndromes or disorders. The following are examples of metabolic disorders that arise from the abnormalities in the enzyme function:

1.   Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency

Glucose-6-Phosphate Dehydrogenase, abbreviated as G6PD, is a rate-limiting enzyme in the pentose phosphate pathway that converts glucose-6-phosphate into 6-phosphoglucono-δ-lactone. This reaction is essential in the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) which protects the cells against oxidative stress. Mutations in the gene encoding the enzyme G6PD cause dysfunctional G6PD activity which affects the accumulation and the stability of NADPH. Red blood cells, which are unable to produce other proteins and acquire NADPH from other unaffected metabolic pathways, are especially sensitive to the reduced NADPH level. G6PD patients are at risk of hemolytic anemia, which occurs when the breakdown of the red blood cells is faster than the rate of replenishment. It can be triggered by certain bacterial or viral infections, certain drugs, or after exposure to fava beans or their pollen.[8]

2.   Gaucher Disease

Gaucher disease is a rare genetic disease caused by mutations in the GDA1 gene, leading to the reduction in the activity of glucocerebrosidase, a lysosomal enzyme that hydrolyzes glucosylceramide into ceramide and glucose. Patients suffering from Gaucher disease are unable to degrade glucosylceramides and other glycolipids, and they are instead deposited in various organs. Clinical manifestations of Gaucher disease can range from mild movement disorders to severe neurological disorders and multi-organ chronic disorders.[9]

In Conclusion

Enzymes catalyze reactions by decreasing the activation energy, stabilizing the transition state and accelerating the rate of equilibrium. They are classified into six groups based on the type of their catalytic reactions, and impairments in certain enzyme activity can lead to metabolic disorders.

All in all, enzymes are key to the regulation of metabolism which sustains life and dictates how cells respond to internal and external stimuli. The active site of an enzyme serves as a catalytic center, and its structure determines the substrate specificity. Both are pivotal to the compatibility between the enzyme, its substrate and the biochemical reaction.

References

  1. Voet D, Voet JG and Pratt CW, Fundamentals of Biochemistry, 2nd edition. New Jersey: John Wiley & Sons; 2006. Chapter 11 Enzyme Function I: Catalysis.
  2. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Protein Function. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26911/
  3. Boyer R, Concepts in Biochemistry, 3rd edition. New Jersey: John Wiley & Sons; 2006. Chapter 5 Enzymes I: Kinetics, Mechanisms and Inhibitions.
  4. Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 2.5, Activation Energy and Reaction Rate. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21680/
  5. Appleby CA and Morton RK. Lactic dehydrogenase and cytochrome b2 of baker yeast; purification and crystallization. Biochem J. 1959 Mar;71(2):492-9.
  6. Zhang Z, Tian C, Zhou S, et al. Mechanism-based design, synthesis and biological studies of N5-substituted tetrahydrofolate analogs as inhibitors of cobalamin-dependent methionine synthase and potential anticancer agents. European Journal of Medicinal Chemistry. 58: 228-36.
  7. Lobell M. and Crout DH. Pyruvate Decarboxylase: A Molecular Study of Pyruvate Decarboxylation and Acylate Formation. J. Am. Chem Soc. 118(8): 1867-1873.
  8. Au SW, Gover S, Lam VM, et al. Human glucose-6-phosphate dehydrogenases: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency. Structure. 2000 Mar;8(3): 292-303. doi: https://doi.org/10.1016%2FS0969-2126%2800%2900104-0
  9. Stirnemann J, Belmatoug N, Camou, F, et al. A Review of Gaucher Disease Pathophysiology, Clinical Presentation and Treatment. Int J Mol Sci. 2017 Feb; 18(2). doi: https://dx.doi.org/10.3390%2Fijms18020441

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