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FACTORS AFFECTING ENZYME ACTIVITY

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Nafeesa Naeem

Enzymes are proteins that catalyze chemical reactions and increase reaction rates. Several factors affect enzyme function, including enzyme concentration, substrate concentration, temperature, pH, inhibitors, activators, and salinity. Enzyme activity is highest when conditions are optimal for maintaining the enzyme's three-dimensional structure. Temperature and pH that are too high or low can cause enzymes to denature by disrupting bonds and changing their shape. Inhibitors also decrease reaction rates by competing with substrates or altering the enzyme's structure.

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Enzymes Enzymes are proteins that speed up chemical reactions. They are needed to do all sorts of making energy. Enzymes are like machines that make tasks easier for your cells. Just like a well-oiled machine works better than a rusty one, certain factors can make enzymes work better or worse. Factors Affecting Enzyme Function  Enzyme concentration  Substrate concentration  Temperature  pH  Inhibitors  Activators  Allosteric Factors  Salinity Enzyme Concentration In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. Graphically this can be represented as: Fig# 01 ‘Zero Order’ reaction rate is independent of substrate concentration These reactions are said to be "zero order" because the rates are independent of substrate concentration, and are equal to some constant k. The formation of product proceeds at a rate which is linear with time. The addition of more substrate does not serve to increase the rate. In zero order kinetics, allowing the assay to run for double time results in double the
amount of product. 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 Table I Reaction Orders with Respect to_ Substrate Concentration Order Rate Equation Comments zero rate = k rate is independent of substrate concentration first rate = k[S] rate is proportional to the first power of substrate concentration second rate = k[S] [S]=k[S]2 rate is proportional to the square of the substrate concentration rate is proportional to the first second rate = k[S1][S2] power of each of two reactants An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied only when the reaction is zero order. When the concentration of the product of an enzymatic reaction is plotted against time, a similar curve results, Figure 02 Fig# 02 Reaction rate limited by substrate concentration
Between A and B, the curve represents a zero order reaction; that is, one in which the rate is constant with time. As substrate is used up, the enzyme's active sites are no longer saturated, substrate concentration becomes rate limiting, and the reaction becomes first order between B and C. To measure enzyme activity ideally, the measurements must be made in that portion of the curve where the reaction is zero order. A reaction is most likely to be zero order initially since substrate concentration is then highest. To be certain that a reaction is zero order, multiple measurements of product (or substrate) concentration must be made. Substrate Concentration It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity (delta A/delta T). This is represented graphically in Figure 3 Fig# 03 Effect of Substrate concentration It is theorized that when this maximum velocity had been reached, all of the available enzyme has been converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax. Using this maximum velocity and equation 7. Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of reaction speed from measurable laboratory data. The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity. This is shown in Figure 3. Using this constant and the fact that Km can also be defined as:
K+1, K-1 and K+2 being the rate constants from equation (7) . Michaelis developed the following expression for the reaction velocity in terms of this constant and the substrate concentration. The size of Km tells us several things about a particular enzyme. 1. A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations. 2. A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity. 3. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes. Temperature Increasing temperature increases the Kinetic Energy that molecules possess. In a fluid, this means that there are more random collisions between molecules per unit time. Since enzymes catalyze reactions by randomly colliding with Substrate molecules, increasing temperature increases the rate of reaction, forming more product. However, increasing temperature also increases the Vibrational Energy that molecules have, specifically in this case enzyme molecules, which puts strain on the bonds that hold them together. As temperature increases, more bonds, especially the weaker Hydrogen and Ionic bonds, will break as a result of this strain. Breaking bonds within the enzyme will cause the Active Site to change shape.
This change in shape means that the Active Site is less complementary to the shape of the Substrate, so that it is less likely to catalyze the reaction. Eventually, the enzyme will become Denatured and will no longer function. As temperature increases, more enzymes' molecules' Active Sites' shapes will be less complementary to the shape of their Substrate, and more enzymes will be denatured. This will decrease the rateof reaction. In summary, as temperature increases, initially the rate of reaction will increase, because of increased Kinetic Energy. However, the effect of bond breaking will become greater and greater, and the rate of reaction will begin to decrease. Fig#04 Effect of Temperature on reaction rate  Optimum T° 1. greatest number of molecular collisions 2. human enzymes = 35°- 40°C 3. body temp = 37°C  Heat: increase beyond optimum T° 1. increased energy level of molecules disrupts bonds in enzyme & between enzyme & substrate 2. H, ionic = weak bonds 3. denaturation = lose 3D shape (3° structure)  Cold: decrease T° 1. molecules move slower 2. decrease collisions between enzyme & substrate
pH - Acidity and Basicity pH measures the Acidity and Basicity of a solution. It is a measure of the Hydrogen Ion (H+) concentration, and therefore a good indicator of the Hydroxide Ion (OH-) concentration. It ranges from pH1 to pH14. Lower pH values mean higher H+ concentrations and lower OH- concentrations. Acid solutions have pH values below 7, and Basic solutions (alkalis are bases) have pH values above 7. Deionised water is pH7, which is termed 'neutral'. H+ and OH- Ions are charged and therefore interfere with Hydrogen and Ionic bonds that hold together an enzyme, since they will be attracted or repelled by the charges created by the bonds. This interference causes a change in shape of the enzyme, and importantly, its Active Site. Different enzymes have different Optimum pH values. This is the pH value at which the bonds within them are influenced by H+ and OH- Ions in such a way that the shape of their Active Site is the most Complementary to the shape of their Substrate. At the Optimum pH, the rate of reaction is at an optimum. Any change in pH above or below the Optimum will quickly cause a decrease in the rate of reaction, since more of the enzyme molecules will have Active Sites whose shapes are not (or at least are less)Complementary to the shape of their Substrate. Fig#5 Effect of pH on reaction rate Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. Changes in pH 1. adds or remove H+ 2. disrupts bonds, disrupts 3D shape 3. disrupts attractions between charged amino acids 4. affect 2° & 3° structure 5. denatures protein The optimum pH value will vary greatly from one enzyme to another, as Table II show:
Table II pH for Optimum Activity Enzyme pH Optimum Lipase (pancreas) 8.0 Lipase (stomach) 4.0 - 5.0 Lipase (castor oil) 4.7 Pepsin 1.5 - 1.6 Trypsin 7.8 - 8.7 Urease 7.0 Invertase 4.0 Effects of Inhibitors on Enzyme Activity Enzyme inhibitors are substances which alter the catalytic action of the enzyme and co nsequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non- competitive and substrate inhibition. Most theories concerning inhibition mechanisms are based on the existence of the enzyme-substrate complex Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. A theory called the "lock -key theory" of enzyme catalysts can be used to explain why inhibition occurs. The lock and key theory utilizes the concept of an "active site." The concept holds that one particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in such a way that its conversion to the reaction products is more favorable. Fig# 06 Lock-Key theory- Competitive Inhibition
If we consider the enzyme as the lock and the substrate the key (Figure 6) - the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds. However, when an inhibitor which resembles the substrate is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds normally. Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate. Figure 7. Fig#07 Non-competitive inhibition Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 08 shows the reaction velocity decreasing after the maximum velocity has been reached. Fig#08 Substrate become rate-inhibiting Allosteric Factors
There are some enzymes which have one active site and one or more regulatory sites and are known as allosteric enzymes. A molecule that binds with the regulatory sites are referred to as allosteric factor. When this molecule in the cellular environment forms a weak non covalent bond at the regulatory site, the shape of the enzyme and its activation center get modified. This change usually decreases the enzyme activity as it inhibits the formation of a new enzyme-substrate complex. However, there are some allosteric activators that promote the affinity between the enzyme and the substrate and influence enzymatic behavior positively. Salinity Salinity may be important not just in solubility, but in promoting enzyme activity. Higher salinity may promote binding of a hydrophobic substrate to an enzyme or of hydrophobic residues to each other within the enzyme to ensure optimal folding for enzymatic activity. Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1- 500 mM. As usual there are exceptions such as the halophilic (salt loving) algae and bacteria. Low salt concentration, the charged amino acid side of the enzyme molecules will attract each other (thus the enzyme become denatured and develop inactive precipitates. High salt concentration of salt, blockage of regular charged group reactions; new interactions will occur; (and again) enzyme precipitates will occur Intermediate salt concentration (like in our blood around 0.9%), is the optimum (favorable outcome) for many enzymes → salt concentration FIG.09 Graph showing the variation of salt concentration with rate of reaction  Salt concentration 1. changes in salinity 2. adds or removes cations (+) & anions (–) 3. disrupts bonds, disrupts 3D shape 4. disrupts attractions between charged amino acids 5. affect 2° & 3° structure 6. denatures protein 7. enzymes intolerant of extreme salinity References:
1. Bennett, T. P., and Frieden, E.: Modern Topics in Biochemistry, pg. 43-45,Macmillan, London (1969). 2. Holum, J.: Elements of General and Biological Chemistry, 2nd ed., 377, Wiley, NY (1968). 3. Martinek, R.: Practical Clinical Enzymology: J. Am. Med. Tech., 31, 162 (1969). 4. Harrow, B., and Mazur, A.: Textbook of Biochemistry, 109, Saunders, Philadelphia (1958). 5. Pfeiffer, J.: Enzymes, the Physics and Chemistry of Life, pg 171-173, Simon and Schuster, NY (1954)
FACTORS AFFECTING ENZYME ACTIVITY

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FACTORS AFFECTING ENZYME ACTIVITY

  • 1. Enzymes Enzymes are proteins that speed up chemical reactions. They are needed to do all sorts of making energy. Enzymes are like machines that make tasks easier for your cells. Just like a well-oiled machine works better than a rusty one, certain factors can make enzymes work better or worse. Factors Affecting Enzyme Function  Enzyme concentration  Substrate concentration  Temperature  pH  Inhibitors  Activators  Allosteric Factors  Salinity Enzyme Concentration In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. Graphically this can be represented as: Fig# 01 ‘Zero Order’ reaction rate is independent of substrate concentration These reactions are said to be "zero order" because the rates are independent of substrate concentration, and are equal to some constant k. The formation of product proceeds at a rate which is linear with time. The addition of more substrate does not serve to increase the rate. In zero order kinetics, allowing the assay to run for double time results in double the
  • 2. amount of product. 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 Table I Reaction Orders with Respect to_ Substrate Concentration Order Rate Equation Comments zero rate = k rate is independent of substrate concentration first rate = k[S] rate is proportional to the first power of substrate concentration second rate = k[S] [S]=k[S]2 rate is proportional to the square of the substrate concentration rate is proportional to the first second rate = k[S1][S2] power of each of two reactants An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied only when the reaction is zero order. When the concentration of the product of an enzymatic reaction is plotted against time, a similar curve results, Figure 02 Fig# 02 Reaction rate limited by substrate concentration
  • 3. Between A and B, the curve represents a zero order reaction; that is, one in which the rate is constant with time. As substrate is used up, the enzyme's active sites are no longer saturated, substrate concentration becomes rate limiting, and the reaction becomes first order between B and C. To measure enzyme activity ideally, the measurements must be made in that portion of the curve where the reaction is zero order. A reaction is most likely to be zero order initially since substrate concentration is then highest. To be certain that a reaction is zero order, multiple measurements of product (or substrate) concentration must be made. Substrate Concentration It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity (delta A/delta T). This is represented graphically in Figure 3 Fig# 03 Effect of Substrate concentration It is theorized that when this maximum velocity had been reached, all of the available enzyme has been converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax. Using this maximum velocity and equation 7. Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of reaction speed from measurable laboratory data. The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity. This is shown in Figure 3. Using this constant and the fact that Km can also be defined as:
  • 4. K+1, K-1 and K+2 being the rate constants from equation (7) . Michaelis developed the following expression for the reaction velocity in terms of this constant and the substrate concentration. The size of Km tells us several things about a particular enzyme. 1. A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations. 2. A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity. 3. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes. Temperature Increasing temperature increases the Kinetic Energy that molecules possess. In a fluid, this means that there are more random collisions between molecules per unit time. Since enzymes catalyze reactions by randomly colliding with Substrate molecules, increasing temperature increases the rate of reaction, forming more product. However, increasing temperature also increases the Vibrational Energy that molecules have, specifically in this case enzyme molecules, which puts strain on the bonds that hold them together. As temperature increases, more bonds, especially the weaker Hydrogen and Ionic bonds, will break as a result of this strain. Breaking bonds within the enzyme will cause the Active Site to change shape.
  • 5. This change in shape means that the Active Site is less complementary to the shape of the Substrate, so that it is less likely to catalyze the reaction. Eventually, the enzyme will become Denatured and will no longer function. As temperature increases, more enzymes' molecules' Active Sites' shapes will be less complementary to the shape of their Substrate, and more enzymes will be denatured. This will decrease the rateof reaction. In summary, as temperature increases, initially the rate of reaction will increase, because of increased Kinetic Energy. However, the effect of bond breaking will become greater and greater, and the rate of reaction will begin to decrease. Fig#04 Effect of Temperature on reaction rate  Optimum T° 1. greatest number of molecular collisions 2. human enzymes = 35°- 40°C 3. body temp = 37°C  Heat: increase beyond optimum T° 1. increased energy level of molecules disrupts bonds in enzyme & between enzyme & substrate 2. H, ionic = weak bonds 3. denaturation = lose 3D shape (3° structure)  Cold: decrease T° 1. molecules move slower 2. decrease collisions between enzyme & substrate
  • 6. pH - Acidity and Basicity pH measures the Acidity and Basicity of a solution. It is a measure of the Hydrogen Ion (H+) concentration, and therefore a good indicator of the Hydroxide Ion (OH-) concentration. It ranges from pH1 to pH14. Lower pH values mean higher H+ concentrations and lower OH- concentrations. Acid solutions have pH values below 7, and Basic solutions (alkalis are bases) have pH values above 7. Deionised water is pH7, which is termed 'neutral'. H+ and OH- Ions are charged and therefore interfere with Hydrogen and Ionic bonds that hold together an enzyme, since they will be attracted or repelled by the charges created by the bonds. This interference causes a change in shape of the enzyme, and importantly, its Active Site. Different enzymes have different Optimum pH values. This is the pH value at which the bonds within them are influenced by H+ and OH- Ions in such a way that the shape of their Active Site is the most Complementary to the shape of their Substrate. At the Optimum pH, the rate of reaction is at an optimum. Any change in pH above or below the Optimum will quickly cause a decrease in the rate of reaction, since more of the enzyme molecules will have Active Sites whose shapes are not (or at least are less)Complementary to the shape of their Substrate. Fig#5 Effect of pH on reaction rate Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. Changes in pH 1. adds or remove H+ 2. disrupts bonds, disrupts 3D shape 3. disrupts attractions between charged amino acids 4. affect 2° & 3° structure 5. denatures protein The optimum pH value will vary greatly from one enzyme to another, as Table II show:
  • 7. Table II pH for Optimum Activity Enzyme pH Optimum Lipase (pancreas) 8.0 Lipase (stomach) 4.0 - 5.0 Lipase (castor oil) 4.7 Pepsin 1.5 - 1.6 Trypsin 7.8 - 8.7 Urease 7.0 Invertase 4.0 Effects of Inhibitors on Enzyme Activity Enzyme inhibitors are substances which alter the catalytic action of the enzyme and co nsequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non- competitive and substrate inhibition. Most theories concerning inhibition mechanisms are based on the existence of the enzyme-substrate complex Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. A theory called the "lock -key theory" of enzyme catalysts can be used to explain why inhibition occurs. The lock and key theory utilizes the concept of an "active site." The concept holds that one particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in such a way that its conversion to the reaction products is more favorable. Fig# 06 Lock-Key theory- Competitive Inhibition
  • 8. If we consider the enzyme as the lock and the substrate the key (Figure 6) - the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds. However, when an inhibitor which resembles the substrate is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds normally. Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate. Figure 7. Fig#07 Non-competitive inhibition Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 08 shows the reaction velocity decreasing after the maximum velocity has been reached. Fig#08 Substrate become rate-inhibiting Allosteric Factors
  • 9. There are some enzymes which have one active site and one or more regulatory sites and are known as allosteric enzymes. A molecule that binds with the regulatory sites are referred to as allosteric factor. When this molecule in the cellular environment forms a weak non covalent bond at the regulatory site, the shape of the enzyme and its activation center get modified. This change usually decreases the enzyme activity as it inhibits the formation of a new enzyme-substrate complex. However, there are some allosteric activators that promote the affinity between the enzyme and the substrate and influence enzymatic behavior positively. Salinity Salinity may be important not just in solubility, but in promoting enzyme activity. Higher salinity may promote binding of a hydrophobic substrate to an enzyme or of hydrophobic residues to each other within the enzyme to ensure optimal folding for enzymatic activity. Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1- 500 mM. As usual there are exceptions such as the halophilic (salt loving) algae and bacteria. Low salt concentration, the charged amino acid side of the enzyme molecules will attract each other (thus the enzyme become denatured and develop inactive precipitates. High salt concentration of salt, blockage of regular charged group reactions; new interactions will occur; (and again) enzyme precipitates will occur Intermediate salt concentration (like in our blood around 0.9%), is the optimum (favorable outcome) for many enzymes → salt concentration FIG.09 Graph showing the variation of salt concentration with rate of reaction  Salt concentration 1. changes in salinity 2. adds or removes cations (+) & anions (–) 3. disrupts bonds, disrupts 3D shape 4. disrupts attractions between charged amino acids 5. affect 2° & 3° structure 6. denatures protein 7. enzymes intolerant of extreme salinity References:
  • 10. 1. Bennett, T. P., and Frieden, E.: Modern Topics in Biochemistry, pg. 43-45,Macmillan, London (1969). 2. Holum, J.: Elements of General and Biological Chemistry, 2nd ed., 377, Wiley, NY (1968). 3. Martinek, R.: Practical Clinical Enzymology: J. Am. Med. Tech., 31, 162 (1969). 4. Harrow, B., and Mazur, A.: Textbook of Biochemistry, 109, Saunders, Philadelphia (1958). 5. Pfeiffer, J.: Enzymes, the Physics and Chemistry of Life, pg 171-173, Simon and Schuster, NY (1954)