Enzymes Factors that Affect Enzyme Activity Several conditions can alter enzyme activity, including changes in temperature

a nd pH and changes in the concentration of substrate and other molecules that bin d to enzymes. The activity of most enzymes is strongly altered by changes in pH and temperatur e. Characteristically, enzymes reach maximal activity within a narrow range of temperature or pH; at levels outside this range, enzyme activity drops off. Effects of Temperature on Enzyme Activity Temperature has a general effect on chemical reactions of all kinds. As the tem perature rises, the rate of chemical reactions typically increases. Due to this there are increases in the kinetic motion of all molecules, with more frequent and stronger collisions as the temperature rises. Temperature also has an effe ct on all proteins, including enzymes. As the temperature rises, the kinetic mo tions of the amino acid chains of an enzyme increases, along with the strength a nd frequency of collisions between enzymes and surrounding molecules. At some p oint, these disturbances become strong enough to denature the enzyme: the hydrog en bonds and other forces that maintain its 3-D structure break, making the enzy me unfold and lose its function. In the range of 0oC to about 40oC, the reaction rate doubles for every 10oC incr ease in temperature. Above 40oC, the increasing kinetic motion begins to unfold the enzyme, reducing the rate of increase in enzyme activity. At some point, a s temperature continues to rise, the unfolding causes the reaction rate to level off at a peak. Further increases cause such extensive unfolding that the react ion rate decreases rapidly to zero. For most enzymes, the peak in activity or o ptimum temperature lies between 40oC and 50oC. Environmental temperature on enzyme activity in Siamese cats ears, nose, paws and tail contains more dark brown pigmen The fur on the extremities t (melanin) than the rest of the body. A heat-sensitive enzyme controlling mela nin production is denatured in warmer body regions, so dark pigment is not produ ced and fur colour is lighter. Effect of pH on enzyme activity Each enzyme has an optimal pH where it operates at peak efficiency in speeding t he rate of its biochemical reaction. On either side of this pH optimum, the rat e of the catalyzed reaction decreases because of the resulting alterations in ch arged groups. The effects on the structure and function of the active site beco me more extreme at pH values farther from the optimum, until the rate drops to z ero. Most enzymes have an optimum pH near that of the cellular contents, about pH 7. Any increase or decrease in pH usually results in the breaking of the hydrogen bonds that help keep the 3-D shape of the enzyme. This will essentially denatur e the enzyme and cause the reaction to cease. However, there are some enzymes t hat work best at more acidic or alkaline conditions. For example, pepsin is a p rotein-digesting enzyme secreted in the stomach and it works best at pH 1.5. Tr ypsin is also a protein-digesting enzyme secreted in the duodenum which works be st at pH 8. Effect of substrate concentration on enzyme activity When substrate concentration is altered in an experiment from low to high and th e temperature and concentration of enzyme molecules are held constant, the rate of enzyme catalysis eventually levels off. At very low concentrations, substrat e molecules collide so infrequently with enzyme molecules that the reaction proc eeds slowly. As the substrate concentration increases, the reaction rate initia lly increases as enzyme and substrate molecules collide more frequently. Howeve r, as the enzyme molecules approach the maximum rate at which they can combine w ith reactants and release products, increasing substrate concentration has a sma

ller and smaller effect and the rate of reaction eventually levels off. When th e enzymes are cycling as rapidly as possible, further increases in substrate con centration have no effect on the reaction rate. At this point, the enzymes are said to be saturated, and the reaction rate remains constant at the saturation l evel (horizontal dashed line). (a) As the substate concentration is increased the rate of reaction increases. There are more collisions between the substrate and the enzyme such that more ac tivated complex's are formed and therefore product per unit time. (b) Further increases in substrate also increase the rate but proportionately le ss than previously. The number of occupied active site is increasing and there is competition for th e active site. (c) The rate is constant. The enzyme active site is fully saturated with substrate such that adding more s ubstrate does not increase the rate of reaction. The enzymes molecules are fully occupied converting substrate to product and any substrate must await a free ac tive site before conversion to product. Effect of enzyme concentration on enzyme activity Given that the substrate concentration is maintained at a high level, and other conditions such as pH and temperature are kept constant, the rate of reaction is proportional to the enzyme concentration. As the enzyme concentration is incre ased, so will the rate of the enzyme reaction. Enzyme Inhibitors The rate at which enzymes catalyze reactions may be reduced or inhibited by vari ous substances. These substances are known as enzyme inhibitors. Some inhibito rs work by binding to the active site of an enzyme while others combine with cri tical sites located elsewhere in the structure of an enzyme. Inhibition may be competitive or non-competitive. Non-competitive inhibition may be reversible or irreversible. Competitive Inhibition In this type of inhibition there are substances with a close structural resembla nce to the substrate of the enzyme. This enables them to be able to fit into th e active site of the enzyme. These substances are known as competitive inhibito rs. As the inhibitor is not acted on by the enzyme it may then remain bound to it, excluding substrate molecules from the active site whilst it remains attache d. In this way it competes for the active site of the enzyme. However, if the substrate concentration is raised to a sufficiently high level, the inhibitor is progressively replaced at the active sites by the substrate. Example 1 The action of the inhibitor malonate on the enzyme succinate dehydrogenase (pg. 124, Fig. 4.12) The enzyme is important to the Krebs Cycle in respiration. Without inhibitor: Succinate dehydrogenase + succinate = Fumarate With inhibitor: Succinate dehydrogenase + malonate = No product Example 2 Treatment of persons who have drunk ethylene glycol (antifreeze) Ethylene glycol is rapidly converted to oxalic acid which can cause irreversible kidney damage. Competition occurs in the presence of ethanol. If the poisone d person is given a large dose of ethanol, the ethanol acts as a competitive inh ibitor, slowing down the action of the enzyme on the antifreeze long enough for it to be excreted. Example 3 Inhibition of aldehyde oxidase by the drug Antabuse (used to help alcoholics)

Ethanol is metabolized in the body by oxidation to acetaldehyde, which is in tur n further oxidized to acetic acid by aldehyde oxidase enzymes. Normally, the se cond reaction is rapid so that acetaldehyde does not accumulate in the body. A drug, disulfiram (Antabuse) inhibits the aldehyde oxidase which causes the accum ulation of acetaldehyde with subsequent unpleasant side-effects of nausea and vo miting. This drug is sometimes used to help people overcome the drinking habit. Non-competitive Inhibition Reversible Inhibition The inhibitors in this type of action have no structural similarity to the subst rate molecule. It binds to the enzyme at some other point at or near its active site. It does not affect the ability of the substrate to bind with the enzyme but makes catalysis impossible to take place. They reduce the rate of reaction and cause the enzyme to work less effectively. When inhibition saturation is re ached the rate of reaction will almost be nil. However, unlike competitive inhi bition an increase in substrate concentration will not increase the rate of reac tion. Example 1 Cyanide poisoning Cyanide is an extremely effective reversible inhibitor of cytochrome oxidase. A concentration of 1 mM KCN (potassium cyanide) is sufficient to inhibit oxygen c onsumption by mitochondria from a vertebrate source by >98%. It attaches to the copper prosthetic group of cytochrome oxidase where it inhibits respiration by preventing the removal of H atoms at the end of the respiratory chain by O2. Th e H atoms will accumulate and aerobic respiration will cease. The H atoms would be combined with the O2 to form water. With the inhibition of the enzyme, ATP is also prevented from being made. (Some scientists class cyanide as an irreversible inhibitor. Regardless of whic h heading it is under it is a non-competitive inhibitor that affects humans and other animals because of it action which is almost instant.) Irreversible Inhibition Irreversible inhibitors bind tightly and permanently to enzymes and destroy thei r catalytic properties. These inhibitors may act at, near, or remote from the a ctive site. Consequently, they may not be displaced by the addition of excess s ubstrate. These effects occur at very low concentrations of the inhibitor and c an be described as poisons. Since many enzymes contain sulphydryl (-SH), alcohol, or acid groups as part of their active sites, any chemical which can react with them acts as an irreversib le inhibitor. Very small concentrations of chemical reagents such as the heavy metal ions mercury (Hg2+), silver (Ag+), arsenic (As+) and lead (Pb2+) or certai n iodide-containing compounds completely inhibit some enzymes. These ions have strong affinities for -SH groups and will combine with these groups, forming str ong covalent bonds. These sulphydryl groups are integral to maintaining the sha pe of the protein and can be found at the active site or elsewhere on the enzyme . These changes in the structure of the enzymes make them ineffective as cataly sts. Example 1 Nerve gas (Sarin) Sarin is a nerve gas that caused the death of several persons and injured many o thers when released by terrorists in the Tokyo subway in 1995. It is a small mo lecule that binds covalently to the R group on the amino acid serine, which is f ound in the active site of the enzyme acetylcholinesterase. This enzyme is very important in the nervous system. With the enzyme inhibited, acetylcholine buil ds up in the synapse and continues to act so that any nerve impulses are, in eff ect, continually transmitted. Normally, the acetylcholinesterase breaks down th e acetylcholine in the synaptic cleft in order to allow the effector muscle or o rgan to relax. Initial symptoms following exposure to sarin are a runny nose, tightness in the

chest and constriction of the pupils. Soon after, the victim has difficulty bre athing and experiences nausea and drooling. As the victim continues to lose con trol of bodily functions, the victim vomits, defecates and urinates. This phase is followed by twitching and jerking. Ultimately, the victim becomes comatose and suffocates in a series of convulsive spasms. Example 2 Pesticides The pesticides DDT and parathion are also key inhibitors of the certain enzymes in the nervous system. They act similarly to Sarin. Example 3 Antibiotics Many antibiotics are inhibitors of specific enzymes in bacteria. Penicillin, a toxin made by a fungus, blocks the active site of an enzyme that many bacteria u se to make their cell walls. Without complete cell walls, the bacteria burst an d die. Rate of Reaction You can tell the difference between competitive and non-competitive inhibitors b y their effects in enzyme controlled reactions in which the substrate concentrat ion is increased. At low concentrations of substrate both inhibitors slow the r ate of reaction. At higher substrate concentration the rate of reaction will in the presence of a non-competitive inhibitor does not reach the maximum rate. T his is because the non-competitive inhibitor keeps some of the enzyme out of action In effect the enzyme concentration is lowered. Meanwhile, a competitive inhibit or is largely replaced at the active site by the correct substrate molecule when the substrate is at a high concentration.

Allosteric Regulation by Enzymes In many cases, the molecules that naturally regulate enzyme activity in a cell b ehave something like reversible non-competitive inhibitors. These regulatory mo lecules change an enzyme s shape and the functioning of its active site by binding t o a site (allosteric site) elsewhere on the molecule via non-covalent bonds. Al losteric regulation is the term used to describe any case in which a protein s funct ion at one site is affected by the binding of a regulatory molecule to a separat e site. It may result in either inhibition or stimulation of an enzyme s activities . Enzymes controlled by allosteric regulation typically have two alternate states controlled from the allosteric site. In one state, this is the active form, the enzyme bind strongly to its substrate. In the other state which is the inactiv e form, the enzyme binds weakly or not at all to its substrate. Binding with th e regulatory substances may induce either state. Binding an allosteric inhibito r converts an allosteric enzyme from the active state to the inactive state, whi le binding an allosteric activator converts the enzyme from its inactive state t o its active state. Pg. 125, Fig. 4.14

End-Product inhibition or Feedback Inhibition Frequently, allosteric inhibitors are a product of the metabolic pathway that th ey regulate. If the product accumulates in excess, its effect as an inhibitor a utomatically slows or stops the enzymatic reaction producing it, typically by in hibiting the enzyme that catalyzes the first reaction of the pathway. If the pr oduct becomes too scarce, the inhibition is reduced and its production increases . Regulation of this type, in which the product of a reaction acts as a regulat or of the reaction is termed end-product inhibition or feedback inhibition. Fee dback inhibition prevents cellular resources from being wasted in the synthesis

of molecules made at intermediate steps of the pathway. Pg. 125, Fig. 4.15 Example 1 The enzyme phosphofructokinase catalyses the phosphorylation of fructose phospha te by ATP to give fructose bisphosphate when ATP is at a high concentration (e.g . when mitochondria are actively producing it) it inhibits this enzyme and glyco lysis is inhibited. When ATP starts to be used up in the cell, (e.g. as it is w hen muscles are contracting) its concentration falls and glycolysis is no longer inhibited. Check out this website: http://click4biology.info/c4b/7/pro7.6.htm#five