CRP 201: ENZYMES – LECTURE NOTE 5

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INTRODUCTION

An enzyme is a molecule that catalyses or greatly speeds up the rate at which a chemical reaction will occur. In most cases, enzymes are proteins, but some enzymes, called ribozymes, are made of RNA. The key aspect of an enzyme is that it reduces the activation energy required for a reaction. In doing so, it speeds up the reaction. The factor of speedup can be very large indeed, up to 1020 in some cases. From this we can see that enzymes are vital in biological processes. Without enzymes, many chemical reactions in the cell which make life possible would probably not even occur.

Characteristics of an enzyme.

  1. They speed up the rate of chemical reaction
  2. Enzymes reduce the activation energy.
  3. Enzymes are specific in nature in the type of reactions they catalyse.
  4. They have high molecular weight that range between 12,000 – 1 million.
  5. Enzyme undergoes physical transformation but revert to their original form at the end of the reaction.
  6. They possess active sites which are pocket or cleft that contains amino-acid side chains that participates in substrate binding and catalyses.

We call molecules that participate in a reaction moderated by an enzyme reactants or substrates. We will use both terms interchangeably. The end products of the reaction are called products. During a reaction, the enzyme binds to one or more reactants, and it is the binding of the enzyme to the reactant that lowers the activation energy. It is truly amazing and fortuitous that this process occurs in nature allowing life to exist. We call the site to which a substrate binds to the enzyme the active site.

 

How do enzymes speed up the rate of chemical reaction?

Enzymes speed up the rate of chemical reaction by lowering the activation energy. The activation energy is the barrier that a reaction must overcome before it can proceed to products. The most useful criterion for predicting the spontaneity of a process is the free energy, which is indicated by the symbol G. The value of the change in free energy , G (where the symbol indicates change), gives the needed information about the spontaneity of the process under consideration.

The free energy of a system decreases in a spontaneous (energy-releasing) process, so G is negative ( G< 0). Such a process is called exergonic, meaning that energy is released. When the change in free energy is positive ( G> 0), the process is nonspontaneous. For a nonspontaneous process to occur, energy must be supplied. Nonspontaneous processes are also called endergonic, meaning that energy is absorbed. For a process at equilibrium, with no net change in either direction, the change in free energy is zero ( G = 0). The key point is that processes that release energy are favored. Conversely, processes that require energy are disfavoured. The change in energy depends only on the state of the molecules present at the start of the process and the state of those present at the end of

the process.

The activation energy profile shows the intermediate stages of a reaction, tho se between the initial and final states. Activation energy profiles are essential in the discussion of catalysts. The activation energy directly affects the rate of reaction, and the presence of a catalyst speeds up a reaction by changing the mechanism and thus lowering the activation energy. At the maximum of the curve connecting the reactants and the products lies the transition state with the necessary amount of energy and the correct arrangement of atoms to produce products. The activation energy can also be seen as the amount of free energy required to bring the reactants to the transition state

Factors influencing enzyme activity

When you think of enzymes think of speeding up a reaction. How well an enzyme works determines how much it can speed up a reaction. There are several factors that can influence how well an enzyme works. These are • Temperature

  • pH of the local environment
  • Concentration of the substrate
  • Presence of inhibitors

• Presence of cofactors and coenzymes

Fig 5.1. An illustration of the activation energy and change in free energy in a reaction.

Fig 5.2. An enzyme lowers the activation energy for a reaction.

Temperature: Temperature affects the rate of reaction by speeding it up through increase in disorderliness of molecules. This ensures increase contact of substrate with enzymes. However, the rate at which increase temperature increase the rate of reaction decreases as the temperature gets to a point when it can no longer speed up the rate of the reaction due to denaturation of enzymes.

Concentration of the substrate: Increase concentration of substrate increases the chance of substrate coming in contact with enzymes for catalyses to occur. This can result in increased rate of reaction.

Presence of inhibitors: Inhibitors are molecules that has similar structures as the substrate. They have the ability to occupy the active site of an enzyme and therefore prevent the enzyme from catalysing the substrate.

Fig 5.3. The effect of temperature on enzyme activity.

 

Presence of cofactors and coenzymes: Cofactors are compounds that come in contact with an inactive enzyme in order to make it active. The cofactor is not a protein, but may be some type of inorganic molecule such as a metal ion. Examples include manganese and copper. It is necessary for the cofactor to bind to the enzyme in order for the catalytic reaction mediated by the enzyme to occur. Coenzymes are however, organic molecules and a cofactor but which accept or donate chemical groups.

Types of enzymes

Enzymes can be classified by their mechanism of action or by what they do to the substrates. There are six major classes of enzymes. These include

 

  • Transferase: This type of enzyme transfers a functional group from one substrate to another. Let’s denote the functional group as X and denote the substrates as A and B. Then the reaction catalyzed by a transferase can be characterized as

A – X + B A + B -X

  • Ligase: This type of enzyme joins two molecules together by generating a chemical bond between them. A ligase requires an energy molecule like ATP and the reaction is accompanied by hydrolysis.
  • Isomerase: This type of enzyme, as its name implies, catalyses a spatial rearrangement of the substrate molecule.
  • Lyase: This is an enzyme that catalyses the non-hydrolytic cleavage of single chemical bonds, leaving behind double bonds or a ring structure.
  • Hydrolase: Catalyses the hydrolysis of a chemical bond.
  • Oxidoreductase: catalyses the transfer of one or more electrons from a hydrogen acceptor or electron donor to a hydrogen donor. A reaction of this type is A+ B A + B

 

 

Mechanisms of enzyme catalysed reactions

Enzyme catalysed a reaction by coming in contact with substrates. The point of attachment of substrate to enzyme is the active site. The active sites as earlier stated are pockets or cleft that contains amino-acid side chains that participates in substrate binding and catalyses.

Two important models have been developed to describe the binding process. The first, the lock-andkey model, assumes a high degree of similarity between the shape of the substrate and the geometry of the binding site on the enzyme (Figure 5.4a). The substrate binds to a site whose shape complements its own, like a key in a lock or the correct piece in a three-dimensional jigsaw puzzle. This model has intuitive appeal but is now largely of historical interest because it does not take into account an important property of proteins, namely their conformational flexibility. The second model takes into account the fact that proteins have some three-dimensional flexibility. According to this induced-fit model, the binding of the substrate induces a conformational change in the enzyme that results in a complementary fit after the substrate is bound (Figure 5.4b).

 

 

Fig 5.4b In the induced-fit model, the enzyme

Fig 5.4a. In the lock-and-key model, the shape of the undergoes a conformational change upon binding to substrate and the conformation of the active site are substrate. The shape of the active site becomes

complementary to one another.

. complementary to the shape of the substrate only afterthe substrate binds to the enzyme.

 

 

 

 

 

 

Fig 5.5a. Formation of product from substrate (bound to the enzyme), followed by release of the product.

 

 

Specificity of an enzyme

 

How specific an enzyme is to a given substrate can be classified in one of three ways. These are • Absolute: In this case, an enzyme only works on one substrate producing one product.

  • Relative: If an enzyme has relative specificity, it will work with several structurally similar substrates, catalysing reactions producing structurally similar products.
  • Stereospecific: In this case, the enzyme will only work with one stereoisomer.

For example, D-glucose can serve as a substrate but L-glucose will not.

Enzyme inhibition

An inhibitor, as the name implies, is a substance that interferes with the action of an enzyme and slows the rate of a reaction. A good deal of information about enzymatic reactions can be obtained by observing the changes in the reaction caused by the presence of inhibitors. Inhibitors can affect an enzymatic reaction in two ways. A reversible inhibitor can bind to the enzyme and subsequently be released, leaving the enzyme in its original condition. An irreversible inhibitor reacts with the enzyme to produce a protein that is not enzymatically active and from which the original enzyme cannot be regenerated.

Two major classes of reversible inhibitors can be distinguished on the basis of the sites on the enzyme to which they bind. One class consists of compounds very similar in structure to the substrate. In this case, the inhibitor can bind to the active site and block the substrate’s access to it. This mode of action is called competitive inhibition because the inhibitor competes with the substrate for the active site on the enzyme. Another major class of reversible inhibitors includes any inhibitor that binds to the enzyme at a site other than the active site and, as a result of binding, causes a change in the structure of the enzyme, especially around the active site. The substrate is still able to bind to the active site, but the enzyme cannot catalyse the reaction when the inhibitor is bound to it.

This mode of action is called non-competitive inhibition

Inhibitors can act by competitive, noncompetitive, or uncompetitive inhibition in three ways:

  • Competitive inhibition occurs if an increase in the concentration of the substrate is correlated with a decrease in the degree of inhibition. A competitive inhibitor has a similar chemical structure to the substrate allowing it to bind to the active site of the enzyme. This means that in competitive inhibition, the enzyme can bind to the substrate or the inhibitor. If the enzyme binds the substrate, it cannot bind the inhibitor, explaining why an increase in the concentration of the substrate is associated with a decrease in the degree of inhibition. Also note that increasing the amount of substrate can overcome the effect of the inhibitor and restore the increased rate of the catalysed reaction.
  • Non-competitive inhibition occurs if the degree of inhibition is not affected by a change in the concentration of the substrate. In this case, the inhibitor can bind to the enzyme, or to the enzyme-substrate complex. The inhibitor does not bind to the same site as the substrate does, so the substrate can still bind to the enzyme in the presence of the inhibitor. This explains why the degree of inhibition is not affected by the concentration of the substrate in this case. A noncompetitive inhibitor affects the catalyzed rate of a reaction.
  • Uncompetitive inhibition occurs when an increase in the concentration of the substrate results in an increase in the degree of inhibition.

 

 

Fig 5.6. Modes of action of inhibitors.

The distinction between competitive and noncompetitive inhibitors is that a competitive inhibitor prevents binding of the substrate to the enzyme, whereas a noncompetitive inhibitor does not.

Regulation of enzyme activities

Regulation of enzyme activities relates to how enzyme-catalysed reaction is controlled to avoid excessive or inadequate production of a given product that can result in cell damage. Enzyme activity can be regulated in several ways.

  1. At the level of the chromosome, is the regulation of the expression of the genes that code for the manufacture of enzymes. In this way the amount of enzyme in the cell can be controlled. This is a slow process that acts over the course of hours, days, and weeks.
  2. Enzymes in the cell can be broken down by proteolytic degradation, another slow process.
  3. Enzyme activities can be regulated through allosteric regulations. The activity of some enzymes is regulated by the presence of other molecules. An allosteric site is a site on an enzyme, called allosteric enzymes. other than the active site where molecules can bind. In allosteric regulation, a molecule called an effector binds to the allosteric site. If the effector increases the activity of the enzyme, it is called an allosteric activator. On the other hand, if the effector decreases the activity of the enzyme, it is called an allosteric inhibitor. When the effector binds to the enzyme, it can alter the shape of the active site either enhancing or decreasing the affinity of the enzyme for the substrate. Allosteric enzymes tend to be located at branch points in metabolic pathways, so activating or inhibiting an allosteric enzyme can enhance or retard a given metabolic process. Regulatory enzymes control the rates of biochemical reactions that involve multiple steps. Good examples include glycolysis and the TCA cycle.
  4. Enzyme activities are also regulated through isolation of enzymes or locking them up. That is, the activity of the enzyme is regulated by limiting its access to substrate molecules. An example of this is the lysosome, a structure that restricts the activities of hydrolases by compartmentalizing them in a membrane structure until they are needed.

Coenzymes and cofactors

Cofactors are nonprotein substances that take part in enzymatic reactions and are regenerated for further reaction. Metal ions frequently play such a role, and they make up one of two important classes of cofactors. The other important class (coenzymes) is a mixed bag of organic compounds; many of them are vitamins or are metabolically related to vitamins. If an enzyme is in an inactive form, we call it an apoenzyme. In this case, the enzyme requires the presence of another compound called a cofactor in order to become active. When the enzyme is in the active form, we call it a holoenzyme. That is:

Apoenzyme + Cofactor = Holoenzyme

The cofactor is not a protein, but may be some type of inorganic molecule such as a metal ion. Examples include manganese and copper. It is necessary for the cofactor to bind to the enzyme in order for the catalytic reaction mediated by the enzyme to occur. When the cofactor is an organic molecule, we say that it is a coenzyme. The function of a coenzyme is to accept or donate chemical groups. Unlike an enzyme, a coenzyme is not a protein molecule. Many coenzymes are vitamins or derive from vitamins through metabolic processes (e.g., the B vitamins). If a coenzyme becomes tightly bound to the enzyme, we say it is a prosthetic group. If it is loosely bound to the enzyme, we say it is a co-substrate. Examples of coenzymes to illustrate the role of coenzymes in biological processes.

 

NAD+

A key player in the electron transport chain in mitochondria and in all energy liberating processes within cells is NAD+ or nicotinamide adenine dinucleotide. NAD+ is derived from vitamin B 3 (niacin) in the cell. NAD+ functions as an enzyme cofactor. has three parts—a nicotinamide ring, an adenine ring, and two sugar–phosphate groups linked together. The electron transport reaction in which a pair of electrons is accepted yielding NADH can be written as:

NAD+ + H+ 2e- NADH

In essence, the cofactor NAD+ has accepted the equivalent of two hydride ions H-. This reaction takes place in glycolysis.

FAD

The so-called flavin nucleotides are derived from the Bvitamins and play a role in electron transport. In particular, flavin adenine dinucleotide (FAD) is synthesized from vitamin B2 (riboflavin) and it functions as a prosthetic group for many enzymes where it plays its role in electron transport. In essence, FAD transports electrons by taking on two complete hydrogen atoms in the following reaction:

FAD +2 H+ 2e- FADH2

The type of enzyme which uses FAD as a coenzyme is called a dehydrogenase. FAD becomes linked to a dehydrogenase via a covalent bond.

 

Coenzyme A Fig. 5.7. The structure of nicotinamide

adenine dinucleotide (NAD+)

Coenzyme A plays a role in the oxidation of pyruvate in the citric acid cycle and in the metabolism of fatty acids. When coenzyme A is bound to acetic acid, we call it acetyl-coenzyme A. This molecule is very important in many biochemical reactions. Acetyl-coenzyme A or acetyl Co-A is a compound composed of adenosine diphosphate (ADP), pantothenic acid, and b-mercaptoethylamine. The chemical role of Co-A is as a carrier of acyl groups. This coenzyme plays a fundamental role in the body, the condensation of oxaloacetate with acetyl-CoA forming citric acid is the first step in the citric acid cycle.

Isozymes

When there are two or more different enzymes that have a similar chemical structure and have in general similar taste for substrate molecules we call the group of enzymes isozymes. In a nutshell isozymes are different enzymes that catalyse the same chemical reactions. While they have similar chemical structures, they can behave very differently in the biochemical environment. This is because by changing a few amino acids in the enzyme we can radically alter its enzyme kinetics. So, while two isozymes might have a taste for the same substrate, the strength of that taste may be very different.

 

 

 

Table 1: Examples of coenzymes and the type of reactions they catalyse.

 

 

 

 

 

 

 

 

 

THE END OF CRP 201 LECTURE NOTE 5.

 

 

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