Biochem 107LTOPIC 8: ENZYMES AND DISEASEObjectives: By the end of this session, you should:1. Know what enzymes are and how they catalyze (accelerate the rate of) biochemical reactions. 2. Understand the effects of temperature and pH on enzyme activity. 3. Understand the effects of substrate concentration and enzyme concentration on enzyme activity. 4. Understand the action of several specific enzymes (catalase, papain, pepsin, pancreatic proteases, and amylase), the role of these enzymes in metabolism, and why they are important. BACKGROUND MATERIALGeneral Properties of Enzymes: Many of the characteristic reactions of cellular activity, when taking place in a test tube, proceed either slowly or proceed rapidly only at temperatures and pressures that are incompatible with life. In a living organism, many complex metabolic reactions must proceed at relatively low temperatures and precise rates. The remarkable ability of organisms to carry out chemical reactions that, under ordinary conditions take place extremely slowly, is because of the activity of enzymes. Enzymes are proteins that catalyze biochemical reactions. Like non-protein catalysts, enzymes influence the rate at which a reaction proceeds, but they do NOT affect the overall equilibrium of the reaction. Like other catalysts, enzymes are not themselves consumed in the reactions they catalyze, so they can be used repeatedly. This property means that the concentration of enzymes can be much lower than the concentrations of their substrates. This consideration is not trivial of overallcellular economy. For example, the enzyme catalase catalyzes the conversion oftoxic hydrogen peroxide (H2O2), which is a minor by-product of normal metabolism,to harmless water and molecular oxygen. Each catalase molecule can degrade 44,000 molecules of H2O2 per second, and the catalase molecules can be reutilizedfor this purpose over and over. Imagine the trouble our cells would be in if each molecule of hydrogen peroxide required a separate molecule of catalase for its degradation.In any chemical reaction, before the reacting (substrate) molecules can be converted into product, they must attain enough energy to reach the transition state. In the transition state, the probability is high that the substrate(s) will be converted into product. There are two general ways in which the rate of a chemical reaction can be accelerated: by an increase in temperature or by addition of catalysts. An increase in temperature increases thermal motion and energy, thus increasing the number of molecules capable of reaching the transition state energy level. In many reactions, the reaction rate is approximately doubled for every 10C rise in temperature. The temperature of mammals, however, is very tightly regulated and in fact, reaction rate alterations due tosignificant temperature alterations would play havoc with our normal metabolic controls. Instead, living things use enzymes as biological catalysts.1Biochem 107LEnzymes combine transiently (i.e., not permanently) with their substrates to form a transition complex, often denoted E-S. An enzymatically catalyzed reaction is speeded up because this enzyme-substrate complex provides a reaction route that has a lower energy of activation for the conversion of reactants to products than does the uncatalyzed process. The reaction converting the substrate to theproduct occurs while the substrate is bound to the enzyme. In other words, the enzyme-substrate (E-S)complex is converted to an enzyme-product (E-P) complex that then dissociates into the free enzyme and the product(s) of the reaction. E + S ⇋ ES ⇋ EP → E + PBecause the rate of the reaction is determined by the free energy levels of the rate-limiting transition state (E-S complex) in the reaction pathway, the enzyme allows the reaction to proceed more rapidly (the higher the free energy barriers, the slower the rate). These points are illustrated below by the free energy profiles for a noncatalyzed reaction and the same reaction catalyzed by an enzyme. Note that the free energy levels of substrates and products are the same regardless of the reaction pathway; thus, the net free energy change (-G) of the overall reaction is the same in both cases, i.e., although the rate may increase greatly, the reaction equilibrium is not altered by an enzyme.Some enzymes depend only on their structural features as proteins for catalytic activity, whereas othersrequire non-protein components called cofactors. Cofactors may be metal ions or organic molecules (called coenzymes); some enzymes require both. Cofactors are generally heat-stable, whereas most enzymes lose activity on heating. This loss of activity, called denaturation, comes about because the Energy profiles for the enzymatic and nonenzymatic catalysis of a reaction.2Biochem 107Lspecific three dimensional (tertiary) structure necessary for substrate binding and reaction catalysis is lost when the temperature increases.Rates of enzymatic reactions are influenced by temperature, pH, enzyme concentration, and substrate concentration. These points have been covered in lecture, and are reviewed only briefly below. We will explore some of these factors in our lab exercises today.Influence of Temperature: Enzymes are invariably influenced in a biphasic fashion by temperature. Forshort time periods and within a narrow temperature range, the reaction rate approximately doubles foreach 10 °C rise in incubation temperature (although many enzymes will not tolerate even a 10 °C rise intemperature). As the temperature increases significantly above body temperature, however, the rate decreases, often sharply. This decrease in enzyme activity is caused by heat denaturation of the enzyme, and is nearly always irreversible. Virtually all mammalian enzymes have temperature maxima very close to body temperature (about 37 C), but there are enzymes that can tolerate extreme temperatures (> 100C) and still function fine, e.g., enzymes in bacteria that inhabit hot springs (Yellowstone National Park) or hydrothermal vents (black smokers) at spreading centers on the ocean floor. Influence of pH: Most enzymes have a characteristic pH (actually usually a narrow range of pH) at which their activity is maximal. Above or below this pH, enzyme activity declines. For most enzymes, the optimal pH is a reflection of the composite properties of the enzyme, its