Enzymes are found all around us, they are found in every plant and animal. Any living organism needs enzymes for its functioning. All living being are controlled by chemical reactions. Chemical reactions that are involved in growth, blood coagulation, healing, combating disease, breathing, digestion, reproduction, and everything else are catalyzed by enzymes. Our body contains about 3,000 enzymes that are constantly regenerating, repairing and protecting us.
Enzymes are powerhouses that are able to perform variety of functions in the human body. Enzymes are wondrous chemicals of nature. Enzymes are used in supplement form in medical arena. Although our bodies can make most of the enzymes, our body can wreak havoc the body's enzyme system and cause enzyme depletion due to poor diet, illness, injury and genetics.
Enzymes DefinitionBack to Top
Substrate is the reactant in an enzyme catalyzed reaction. The portion of the molecule that is responsible for catalytic action of enzyme is the active site.
Characteristics of EnzymesBack to Top
- Enzymes possess great catalytic power.
- Enzymes are highy specific.
- Enzymes show varying degree of specificities.
- Absolute specificity where the enzymes react specifically with only one substrate.
- Stereo specificity is where the enzymes can detect the different optical isomers and react to only one type of isomer.
- Reaction specific enzymes, these enzymes as the name suggests reacts to specific reactions only.
- Group specific enzymes are those that catalyze a group of substances that contain specific substances.
- The enzyme activity can be controlled but the activity of the catalysts can not be controlled.
- All enzymes are proteins.
- Like the proteins, enzymes can be coagulated by alcohol, heat, concentrated acids and alkaline reagents.
- At higher temperatures the rate of the reaction is faster.
- The rate of the reaction invovlving an enzyme is high at the optimum temperature.
- Enzymes have an optimum pH range within which the enzymes function is at its peak.
- If the substrate shows deviations larger than the optimum temperature or pH, required by the enzyme to work, the enzymes do not function such conditions.
- Increase in the concentration of the reactants, and substrate the rate of the reaction increase until the enzyme will become saturated with the substrate; increase in the amount of enzyme, increases the rate of the reaction.
- Inorganic substances known as activators increase the activity of the enzyme.
- Inhibitors are substances that decrease the activity of the enzyme or inactivate it.
- Competitive inhibitors are substances that reversibly bind to the active site of the enzyme, hence blocking the substrate from binding to the enzyme.
- Incompetitive inhibitors are substances that bind to any site of the enzyme other than the active site, making the enzyme less active or inactive.
- Irreversible inhibitors are substances that from bonds with enzymes making them inactive.
Enzyme ClassificationBack to Top
The current system of nomenclature of enzymes uses the name of the substrate or the type of the reaction involved, and ends with "-ase". Example:'Maltase'- substrate is maltose. 'Hydrolases'- reaction type is hydrolysis reaction.
Classification of enzymesEnzymes are classified based on the reactions they catalyze into 6 groups: Oxidoreductases, transferases, hydrolases, lyases, isomearses, ligases.
Oxidoreductases - Oxidoreductase are the enzymes that catalyze oxidation-reduction reactions. These emzymes are important as these reactions are responsible for the production of heat and energy.
Transferases - Transferases are the enzymes that catalyze reactions where transfer of functional group between two substrates takes place.
Hydrolases - Hydrolases are also known as hydrolytic enzymes, they catalyze the hydrolysis reactions of carbohydrates, proteins and esters.
Lyases - Lyases are enzymes that catlayze the reaction invvolving the removal of groups from substrates by processes other than hydrolysis by the formation of double bonds.
Isomerases - Isomerases are enzymes that catalyze the reactions where interconversion of cis-trans isomers is involved.
Ligases - Ligases are also known as synthases, these are the enzymes that catalyze the reactions where coupling of two compounds is involved with the breaking of pyrophosphate bonds.
Structure of EnzymesBack to Top
Enzymes are proteins, like the proteins the enzymes contain chains of amino acids linked together. The characteristic of an enzyme is determined by the sequence of amino acid arrangement. When the bonds between the amino acid are weak, they may be broken by conditions of high temperatures or high levels of acids. When these bonds are broken, the enzymes become nonfunctional. The enzymes that take part in the chemical reaction do not undergo permanent changes and hence they remain unchanged to the end of the reaction.
Enzymes are highly selective, they catalyze specific reactions only. Enzymes have a part of a molecule where it just has the shape where only certain kind of substrate can bind to it, this site of activity is known as the 'active site'. The molecules that react and bind to the enzyme is known as the 'substrate'.
Most of the enzymes consists of the protein and the non protein part called the 'cofactor'. The proteins in the enzymes are usually globular proteins. The protein part of the enzymes are known 'apoenzyme', while the non-protein part is known as the cofactor. Together the apoenzyme and cofactors are known as the 'holoenzyme'.
Cofactors may be of three types: prosthetic groups, activators and coenzymes.
Prosthetic groups are organic groups that are permanently bound to the enzyme. Example: Heme groups of cytochromes and bitotin group of acetyl-CoA carboxylase.
Activators are cations- they are positively charged metal ions. Example: Fe - cytochrome oxidase, CU - catalase, Zn - alcohol dehydrogenase, Mg - glucose - 6 - phosphate, etc.
Coenzymes are organic molecules, usually vitamins or made from vitamins. they are not bound permanently to the enzyme, but they combine with the enzyme-substrate complex temporarily. Example: FAD - Flavin Adenine Dinucleotide, FMN - Flavin Mono Nucleotide, NAD - Nicotinamide Adenine Dinucleotide, NADP - Nicotinamide Adenine Dinucleotide.
Function of EnzymesBack to Top
Biological Functions of Enzymes:
- Enzymes perform a wide variety of functions in living organisms.
- They are major components in signal transduction and cell regulation, kinases and phosphatases help in this function.
- They take part in movement with the help of the protein myosin which aids in muscle contraction.
- Also other ATPases in the cell membrane acts as ion pumps in active transport mechanism.
- Enzymes present in the viruses are for infecting cell.
- Enzymes play a important role in the digestive activity of the enzymes.
- Amylases and proteases are enzyme sthat breakdown large molecules into absorbable molecules.
- Variuos enzymes owrk together in a order forming metabolic pathways. Example: Glycolysis.
- Food Processing - Amylases enzymes from fungi and plants are used in production of sugars from starch in making corn-syrup.
- Catalyze enzyme is used in breakdown of starch into sugar, and in baking fermentation process of yeast raises the dough.
- Proteases enzyme help in manufacture of biscuits in lowering the protein level.
- Baby foods - Trypsin enzyme is used in pre-digestion of baby foods.
- Brewing industry - Enzymes from barley are widely used in brewing industries.
- Amylases, glucanases, proteases, betaglucanases, arabinoxylases, amyloglucosidase, acetolactatedecarboxylases are used in prodcution of beer industries.
- Fruit juices - Enzymes like cellulases,pectinases help are used in clarifying fruit juices.
- Dairy Industry - Renin is used inmanufacture of cheese. Lipases are used in ripening blue-mold cheese. Lactases breaks down lactose to glucose and galactose.
- Meat Tenderizes - Papain is used to soften meat.
- Starch Industry - Amylases, amyloglucosidases and glycoamylases converts starch into glucose and syrups.
- Glucose isomerases - production enhanced sweetening properties and lowering calorific values.
- Paper industry - Enzymes like amylases, xylanases, cellulases and liginases lower the viscosity, and removes lignin to soften paper.
- Biofuel Industry - Enzymes like cellulases are used in breakdown of cellulose into sugars which can be fermented.
- Biological detergent - proteases, amylases, lipases, cellulases, asist in removal of protein stains, oily stains and acts as fabric conditioners.
- Rubber Industry - Catalase enzyme converts latex into foam rubber.
- Molecular Biology - Restriction enzymes, DNA ligase and polymerases are used in genetic engineering, pharmacology, agriculture, medicine, PCR techniques, and are also important in forensic science.
List of EnzymesBack to Top
List of enzymes
Examples of EnzymesBack to Top
A few well known examples of enzymes are as follows: Lipases, Amylases, Maltases, Pepsin, Protease, Catalases, Maltase, Sucrase, Pepsin, Renin, Catalases,
A few examples of foods that are rich in enzymes:
Enzymes are available in the food we eat. Foods that are canned, or processed food like irradiation,drying, and freezing make the foods enzyme dead. Refined foods are void of any sort of nutrition. Food that is whole, uncooked and unpasteurized milk will provide enough enzymes. There are two basic ways to increase enzyme intake. First is to eat more fresh foods, cooking tends to kill enzymes. Raw fruits and vegetables are a good source of enzymes. Fermented food like yoghurt, intake improves body's enzyme status. The other way to increase enzyme status of the body is by intake of enzyme supplements.
Here is a list of foods rich in enzymes - Apples, apricots, asparagus, avocado, banana, beans, beets, broccoli, cabbage, carrots, celery, cherries, cucumber, figs, garlic, ginger, grapes, green barley grass,kiwi fruit, etc.
Enzyme kinetics is the study of factors that determine the speed of enzyme-catalysed reactions. It utilizes some mathematical equations that can be confusing to students when they first encounter them. However, the theory of kinetics is both logical and simple, and it is essential to develop an understanding of this subject in order to be able to appreciate the role of enzymes both in metabolism and in biotechnology.
Assays (measurements) of enzyme activity can be performed in either a discontinuous or continuous fashion. Discontinuous methods involve mixing the substrate and enzyme together and measuring the product formed after a set period of time, so these methods are generally easy and quick to perform. In general we would use such discontinuous assays when we know little about the system (and are making preliminary investigations), or alternatively when we know a great deal about the system and are certain that the time interval we are choosing is appropriate.
In continuous enzyme assays we would generally study the rate of an enzyme-catalysed reaction by mixing the enzyme with the substrate and continuously measuring the appearance of product over time. Of course we could equally well measure the rate of the reaction by measuring the disappearance of substrate over time. Apart from the actual direction (one increasing and one decreasing), the two values would be identical. In enzyme kinetics experiments, for convenience we very often use an artificial substrate called a chromogen that yields a brightly coloured product, making the reaction easy to follow using a colorimeter or a spectrophotometer. However, we could in fact use any available analytical equipment that has the capacity to measure the concentration of either the product or the substrate.
In almost all cases we would also add a buffer solution to the mixture. As we shall see, enzyme activity is strongly influenced by pH, so it is important to set the pH at a specific value and keep it constant throughout the experiment.
Our first enzyme kinetics experiment may therefore involve mixing a substrate solution (chromogen) with a buffer solution and adding the enzyme. This mixture would then be placed in a spectrophotometer and the appearance of the coloured product would be measured. This would enable us to follow a rapid reaction which, after a few seconds or minutes, might start to slow down, as shown in Figure 4.
A common reason for this slowing down of the speed (rate) of the reaction is that the substrate within the mixture is being used up and thus becoming limiting. Alternatively, it may be that the enzyme is unstable and is denaturing over the course of the experiment, or it could be that the pH of the mixture is changing, as many reactions either consume or release protons. For these reasons, when we are asked to specify the rate of a reaction we do so early on, as soon as the enzyme has been added, and when none of the above-mentioned limitations apply. We refer to this initial rapid rate as the initial velocity (v0). Measurement of the reaction rate at this early stage is also quite straightforward, as the rate is effectively linear, so we can simply draw a straight line and measure the gradient (by dividing the concentration change by the time interval) in order to evaluate the reaction rate over this period.
We may now perform a range of similar enzyme assays to evaluate how the initial velocity changes when the substrate or enzyme concentration is altered, or when the pH is changed. These studies will help us to characterize the properties of the enzyme under study.
The relationship between enzyme concentration and the rate of the reaction is usually a simple one. If we repeat the experiment just described, but add 10% more enzyme, the reaction will be 10% faster, and if we double the enzyme concentration the reaction will proceed twice as fast. Thus there is a simple linear relationship between the reaction rate and the amount of enzyme available to catalyse the reaction (Figure 5).
This relationship applies both to enzymes in vivo and to those used in biotechnological applications, where regulation of the amount of enzyme present may control reaction rates.
When we perform a series of enzyme assays using the same enzyme concentration, but with a range of different substrate concentrations, a slightly more complex relationship emerges, as shown in Figure 6. Initially, when the substrate concentration is increased, the rate of reaction increases considerably. However, as the substrate concentration is increased further the effects on the reaction rate start to decline, until a stage is reached where increasing the substrate concentration has little further effect on the reaction rate. At this point the enzyme is considered to be coming close to saturation with substrate, and demonstrating its maximal velocity (Vmax). Note that this maximal velocity is in fact a theoretical limit that will not be truly achieved in any experiment, although we might come very close to it.
The relationship described here is a fairly common one, which a mathematician would immediately identify as a rectangular hyperbola. The equation that describes such a relationship is as follows:
The two constants a and b thus allow us to describe this hyperbolic relationship, just as with a linear relationship (y = mx + c), which can be expressed by the two constants m (the slope) and c (the intercept).
We have in fact already defined the constant a — it is Vmax. The constant b is a little more complex, as it is the value on the x-axis that gives half of the maximal value of y. In enzymology we refer to this as the Michaelis constant (Km), which is defined as the substrate concentration that gives half-maximal velocity.
Our final equation, usually called the Michaelis–Menten equation, therefore becomes:
In 1913, Leonor Michaelis and Maud Menten first showed that it was in fact possible to derive this equation mathematically from first principles, with some simple assumptions about the way in which an enzyme reacts with a substrate to form a product. Central to their derivation is the concept that the reaction takes place via the formation of an ES complex which, once formed, can either dissociate (productively) to release product, or else dissociate in the reverse direction without any formation of product. Thus the reaction can be represented as follows, with k1, k−1 and k2 being the rate constants of the three individual reaction steps:
The Michaelis–Menten derivation requires two important assumptions. The first assumption is that we are considering the initial velocity of the reaction (v0), when the product concentration will be negligibly small (i.e. [S] ≫ [P]), such that we can ignore the possibility of any product reverting to substrate. The second assumption is that the concentration of substrate greatly exceeds the concentration of enzyme (i.e. [S]≫[E]).
The derivation begins with an equation for the expression of the initial rate, the rate of formation of product, as the rate at which the ES complex dissociates to form product. This is based upon the rate constant k2 and the concentration of the ES complex, as follows: 1
Since ES is an intermediate, its concentration is unknown, but we can express it in terms of known values. In a steady-state approximation we can assume that although the concentration of substrate and product changes, the concentration of the ES complex itself remains constant. The rate of formation of the ES complex and the rate of its breakdown must therefore balance, where: and
Hence, at steady state:
This equation can be rearranged to yield [ES] as follows: 2
The Michaelis constant Km can be defined as follows:
Equation 2 may thus be simplified to: 3
Since the concentration of substrate greatly exceeds the concentration of enzyme (i.e. [S] ≫ [E]), the concentration of uncombined substrate [S] is almost equal to the total concentration of substrate. The concentration of uncombined enzyme [E] is equal to the total enzyme concentration [E]T minus that combined with substrate [ES]. Introducing these terms to Equation 3 and solving for ES gives us the following: 4
We can then introduce this term into Equation 1 to give: 5
The term k2[E]T in fact represents Vmax, the maximal velocity. Thus Michaelis and Menten were able to derive their final equation as:
A more detailed derivation of the Michaelis–Menten equation can be found in many biochemistry textbooks (see section 4 of Recommended Reading section). There are also some very helpful web-based tutorials available on the subject.
Michaelis constants have been determined for many commonly used enzymes, and are typically in the lower millimolar range (Table 5).
It should be noted that enzymes which catalyse the same reaction, but which are derived from different organisms, can have widely differing Km values. Furthermore, an enzyme with multiple substrates can have quite different Km values for each substrate.
A low Km value indicates that the enzyme requires only a small amount of substrate in order to become saturated. Therefore the maximum velocity is reached at relatively low substrate concentrations. A high Km value indicates the need for high substrate concentrations in order to achieve maximum reaction velocity. Thus we generally refer to Km as a measure of the affinity of the enzyme for its substrate—in fact it is an inverse measure, where a high Km indicates a low affinity, and vice versa.
The Km value tells us several important things about a particular enzyme.
An enzyme with a low Km value relative to the physiological concentration of substrate will probably always be saturated with substrate, and will therefore act at a constant rate, regardless of variations in the concentration of substrate within the physiological range.
An enzyme with a high Km value relative to the physiological concentration of substrate will not be saturated with substrate, and its activity will therefore vary according to the concentration of substrate, so the rate of formation of product will depend on the availability of substrate.
If an enzyme acts on several substrates, the substrate with the lowest Km value is frequently assumed to be that enzyme's ‘natural’ substrate, although this may not be true in all cases.
If two enzymes (with similar Vmax) in different metabolic pathways compete for the same substrate, then if we know the Km values for the two enzymes we can predict the relative activity of the two pathways. Essentially the pathway that has the enzyme with the lower Km value is likely to be the ‘preferred pathway’, and more substrate will flow through that pathway under most conditions. For example, phosphofructokinase (PFK) is the enzyme that catalyses the first committed step in the glycolytic pathway, which generates energy in the form of ATP for the cell, whereas glucose-1-phosphate uridylyltransferase (GUT) is an enzyme early in the pathway leading to the synthesis of glycogen (an energy storage molecule). Both enzymes use hexose monophosphates as substrates, but the Km