Enzymes are considered catalysts; substances that increase the rates of reactions. Enzymes are responsible for thousands of metabolic processes that involve the sustainment of life, one common one is simple food digestion. Without enzymes, digestion would occur too slowly for life to continue. Enzymes maintain a protein structure consisting of one or possibly more than one polypeptide chains of defined primary structure, and take up a characteristic folded form in the native state. Should the structure be modified by an outside entity, the enzyme could be deactivated.
Enzymes, because they are specific with the nature of the reactions they catalyze, they are divided into Stereospecific and chemical specificity. These specificificities allows for better research opportunities and understanding the enzyme.
Stereospecificity is a property of a reaction mechanism that allows for a different stereoisomer reaction product from a different stereoisomeric reactant basically, the enzyme is able to act on a steric or optical isomer. Chemical specificity entails three components: Absolute specificity – the enzyme catalyzes with only one enzyme.
Group specificity – the enzyme will act only on molecules that have specific functional groups. These are more common and such functional groups include: amino, phosphate and methyl groups. Professor explains this by the example: An enzyme catalyzing the hydrolysis of sugar derivatives, such as ß-galactosidase, may require that the sugar be galactose and that this be joined to an aglycone through a ß-linkage to the first C atom of galactose (section 3.2).
Finally, Linkage specificity – this enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure.
It is only concerned with the type of linkage between A and B. For example, an esterase may hydrolyze many esters irrespective of the nature of the alcohol and acid moieties, although this type of specificity is relatively rare (section 3.2). Finally, enzymes aids in understanding the basic enzymatic mechanism and having the ability to select a method for enzyme analysis. Temperature, pH, enzyme inhibitors, substrate concentration, enzyme concentration and allosteric enzymes affect the performance of an enzyme.
Temperature can enhance the rate of enzyme catalyzed reactions. As the temperature rises, the rate of most reactions increases (Tymoczko p. 126). Temperature is important because when temperature increases the kinetic energy of the molecules will also increase, allowing the molecules to move freely. Allowing them to move freely will increase collisions between the enzymes and molecules, thus, more reactions can take place. The rise in temperature increases the Brownian motion of the molecules, which makes interactions between an enzyme and its substrate more likely (Tymoczko p. 126). The high collision rate allows the reaction rate to increase but only up to a certain point.
If the temperature were to increase too much, the enzyme’s protein can begin to weaken and the three dimensional structure is not strong enough to uphold the polypeptide chain’s thermal movement/bumping, causing the protein to lose the structure required for activity. This will cause the protein to denature, a potentially permanent process. Below is a graph of rate against temperature. The temperature with the fastest rate is called optimum temperature. The reaction rate increases with temperature to an optimal, then quickly declines with a further increase of temperature. It should be noted that optimal temperature will vary depending upon how long it has been exposed to the higer temperature this is because many enzxymes are negatively affected by high temperatures.
pH levels can also affect the performance of an enzyme. PH measures the acidity and basicity of a solution. The change of pH levels will affect the polar and non-polar intramolecular attractive and repulsive forces, altering the shape of the enzyme and active site. The active site is the region that binds the substrates and with the interaction of the substrate at the active site, promotion of the formation of the transition state can occur. The substrate locks into the active site of the enzyme. The active site alters its shape holding the substrate more tightly and straining it. An enzyme-substrate complex is formed and the substrate undergoes a chemical change, creating a new product. Once the new product is formed, it is released from the active site (Enzymes).
Enzymes are very large molecules, yet the substances whose reaction they catalyze are usually very small, therefore, if the reactants are to be attached in some way to the protein molecule, they must do so at the active site (Section 3.1). In an acid solution any basic groups such as the Nitrogen groups in the protein would be protonated. Whereas, if the environment was too basic, the acid groups would be deprotonated which would alter the electrical attractions between polar groups. The activity of most enzymes displays a bell-shaped curve when examined as a function of pH (Tymoczko p. 127). The optimal pH, the pH at which enzymes display their greatest activity, varies with the enzyme and is in correlation with the environment of the enzyme. This is due to the fact that the pH can make and break intra- and inter-molecular bonds, changing the shape of the enzyme and the effect of the enzyme. Most enzymes are active only within a narrow pH range usually between 5 and 9. Several factors are influenced directly by the pH in which the reaction takes place:
Some substances decrease or at times can stop the catalytic activity of enzymes in biochemical reactions by blocking or distorting the active site. Such chemicals are known as inhibitors, because they prevent reaction. Thus, enzyme inhibitors can also affect the performance of an enzyme. Inhibition by particular chemicals can be a source of insight into the mechanism of enzyme action (Tymoczko p. 128). Enzyme inhibition can be either reversible or irreversible. Reversible inhibition allows for fast dissociation of the enzyme-inhibitor complex. There are three types of reversible inhibition: competitive, uncompetitive and noncompetitive inhibition. Competitive inhibition occurs when the substrate and a substance resembling the substrate are added to the enzyme. The substrate is prevented from binding to the same active site.
An enzyme can bind substrate, forming an ES complex or inhibitor (EI), but not both (ESI) (Tymoczko p. 128). A competitive inhibitor reduces the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. In order to have a normal reaction take place at a reasonable rate, the concentration of the substrate has to increase causing the substrate to outdo the inhibitor. Penicillin is a competitive inhibitor that blocks the active site of an enzyme that many bacteria use to build their cell walls. Uncompetitive inhibitors indicate the inhibitor binds only to the enzyme-substrate complex and the binding site is created only when the enzyme binds to the substrate (Tymoczko p. 129).
Herbicide glyphosate, or Roundup is an uncompetitive inhibitor in the biosynthetic pathway for aromatic amino acids in plants, the plant dies because it lacks amino acids. In noncompetitive inhibition, the inhibitor does not attach itself to the active site, it attaches elsewhere on the enzyme, and thus it can bind simultaneously to an enzyme at different binding sites. The noncompetitive inhibitor decreases the overall number of active enzymes and unlike competitive inhibition; it cannot overcome by increasing the substrate concentration (Tymoczko p. 129). This results in the changing of the shape and once the shape is changed on the active site, it can no longer be attached to the substrate.
The inhibition of the enzyme, doxycycline prevents the growth and reproduction of bacteria that cause gum disease. In order to determine if a reversible inhibitor acts by competitive, uncompetitive or noncompetitive inhibition one would have to review the Michaelis-Menten kinetics theory. Essentially under this kinetics exhibition, enzymes are not allosterically inhibited. The ability to distinguish between the three types of reversible inhibitions is seen through the measurements of the rates of catalysis at different concentrations of substrate. In competitive inhibition, the inhibitor competes with the substrate for the active site. The major characteristic of competitive inhibition is that it can be overcome by a sufficiently high concentration of substrate (Tymoczko p. 129). In competitive inhibitor, an enzyme will have the same Vmax as in the absence of the inhibitor.
The more inhibitor present, the more substrate is required to displace it and reach Vmax (Tymoczko p. 129). In uncompetitive inhibition, the inhibitor binds only to the ES complex and ESI does not proceed to form any product. In addition, the apparent value of KM will be lowered because the inhibitor binds to ES to form ESI, diminishing ES (Tymoczko p. 129). In noncompetitive inhibition, a substrate can bind to the enzyme-inhibitor complex as we as to the enzyme alone (Tymoczko p. 130). In both cases, the inhibitor-substrate complex does not proceed to form a product.
Substrate concentration is used to describe the number of substrate molecules in a solution. Substrate is considered the substance on which enzymes begin to act. Enzymes are highly specific both in the reactions that they catalyze and in their choice of reactants, which are called substrates (Tymoczko p. 94). During an enzyme reaction, the enzyme combines with the substrate at the active site. As briefly mentioned under the pH section, the enzyme has a special shape that fits exactly with the substrate. An enzyme-substrate complex is formed when the enzyme attaches to the substrate and once the reaction is finished and the product created, they are released from the enzyme, which will then catalyze another reaction.
Substrate concentration affects the performance of an enzyme because when the concentration of substrate increases, the rate of reaction also increases until saturation occurs. There are more collisions between the substrate and the enzyme such that more activated complexes are formed and therefore more product per unit time (Effect of Substrate Concentration). Essentially, as the concentration increases, the rate keeps increasing and once the maximum rate is achieved and there is no free enzyme to bind with, the substrate and all the active sites of enzyme are bound to the substrate. The enzymes molecules are fully occupied converting substrate to product and any other substrate will wait for a free active site before conversion to a product.
Enzyme concentration can also affect the performance of an enzyme. A low enzyme concentration would lead to a reaction that occurs slowly, whereas, a high enzyme concentration can increase the reaction until an optimal rate is achieved. The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship between activity and concentration is affected by many factors such as temperature, pH, etc (Introduction to Enzyme). In order to observe the effect of an enzyme on a reaction, an experiment would take place with low concentrations of the enzyme. The substrate is usually present in large quantities at first. The rate of the reaction can be measured by the amount of product formed over time. As the enzyme solution becomes more concentrated, collisions between enzymes and substrate molecules are more likely to occur.
Therefore, as the enzyme concentration increases, the rate of reaction speeds until it reaches a certain level where it begins to flatten out (Enzymes). The best reaction is when every enzyme on every site is occupied by the substrate. Once this point has been reached, a higher enzyme concentration is required to increase the reaction rate, allowing for new enzymes to become available to bind to the substrate. Once the substrate molecules are attached to the enzymes, increasing the enzyme concentration will not speed up the reaction process. The extra enzymes added will not have any additional substrate to work on and the reaction rate remains level at the maximum limit. The maximum rate for a particular enzyme reaction is Vmax. KM =Michaelis-Menten constant. This constant measures the efficiency of the enzyme. It also describes the variation of enzyme activity as a function of substrate . A low KM indicates the reaction is quick even with low substrate concentrations whereas a high value means the enzyme is not as effective.
Enzymes that regulate the flux of biochemicals through metablic pathways are known as allosteric enzymes. These enzymes change their molecular symmetry upon binding to an effector, allowing for a change in binding affinity at a different binding site. Moreover, these enzymes allow for regulation of catalytic activity and sigmoidla kinetics (Tymoczko p. 112). They do not adhere to the Michaelis-Menten kinetics because they have multiple active sites and multiple subunits. Because of this, they have sigmoidal kinetics and can exist in two states. A sigmoidal plot has an S curve resulting from the combination of the T state and R state curves. State one has R for relaxed and it is the active confirmation which actually catalyzes reactions. The second state, T for tense is less active (Tymoczko p.115).
More enzymes are found in the R state when there is a high concentration and at insufficient substrate amounts, T is the favorite. Essentially both depend on the concentration of the substrate (Tymoczko, p. 68, 6th edition). They are distinctive because they have the ability to adapt to various conditions in the environment. The allosteric inhibitors join with the regulatory site and change the shape of the enzyme.
In allosteric enzymes, the binding of substrate to one active site can affect the properties of other active sites in the same enzyme molecule. An outcome of this interaction between subunits is that the binding of substrate becomes cooperative; the binding of substrate to one active site of the enzyme facilitates substrate binding to the other active sites (Tymoczko, p. 118). In addition, the activity of an allosteric enzyme may be transformed by regulatory molecules that are reversibly bound to specific sites other than the catalytic sites. This allows for the catalytic properties of allosteric enzymes to adjust to meet the immediate needs of a cell. Because of this, allosteric enzymes are essential regulators of metabolic pathways within the cell.
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