GB1_LC_Proteins-Nucleic-Acids-and-Enzymes.docx.pdf

ATENEO DE DAVAO UNIVERSITY Km 7 Central Park Blvd, Talomo, 8016 Davao City, Philippines Tel No. +63 (82) 221.2411 local 8608...

ATENEO DE DAVAO UNIVERSITY Km 7 Central Park Blvd, Talomo, 8016 Davao City, Philippines Tel No. +63 (82) 221.2411 local 8608 E-Mail: [email protected] * www.addu.edu.ph In Consortium with Ateneo de Zamboanga University and Xavier University SENIOR HIGH SCHOOL – GENERAL BIOLOGY 1 Biological Molecules 1 The major categories of Biological molecules are: y ⮚ Carbohydrates ⮚ Proteins og ⮚ Lipids (Fats) ⮚ Nucleic Acids ol All of these molecules exist primarily as organic polymers in living things. A polymer is a molecule that is made up of small repeating units called monomers. Bi Polymer Monomer Carbohydrate Monosaccharide Protein Amino acid al Nucleic Acid Nucleotide er Each of the above categories has different monomers however they all join together the same way through a condensation synthesis reaction. en Furthermore, all of the polymers listed above can break down into their monomers through a hydrolysis reaction. -G Monosaccharides Disaccharides glucose maltose (glucose + glucose) fructose sucrose (glucose + fructose) galactose lactose ( glucose + galactose) U D A. Proteins Ad Structure The monomer of proteins is the amino acid. Amino acids are made up of a central carbon atom attached to a hydrogen atom and 3 groups. Two of these groups are the same for all amino acids: the amine group and the carboxyl group. The last group is different for all of the amino acids; this is commonly referred to as the ‘R’ – group. The generic structure for all amino acids is then:  1 y There are 21 amino acids that are required for building proteins in our bodies. They can og be categorized in two ways: Essential vs. non-essential amino acids ol a. Essential amino acids must be ingested since our bodies do not manufacture these molecules Bi b. Non-essential amino acids can be made by our bodies from other amino acids. Polar vs. nonpolar: This designation has implications about the different levels of structure that can be achieved as we al will note later. er Structure of Proteins Primary Secondary en Tertiary Quaternary -G Functions structural roles - keratin: makes up U hair and nails - collagen: support in ligaments, tendons, and skin D - actin and myosin: make up muscle fibres in muscle cells that allow contraction AND are a major component of the cytoskeleton of cells. Ad - tubulin: hollow cylinders of globular protein arranged in rows to make microtubules; also a major component of the cytoskeleton. They act as highways for organelle movement. - kinesin: protein that moves vesicles/organelles along a microtubule - dynein: protein that moves vesicles/organelles along a microtubule - histones: protein associated with DNA to make chromosomes - intercellular filaments: hold cells together hormonal roles - insulin: messenger molecule in blood from pancreas that signals for cells to absorb glucose. - cyclin: messenger molecule in blood to signal cells to go into stages of mitosis.  transportation roles - hemoglobin: transports O2 in the blood. cell recognition roles - complement system proteins: aid the immune response - antibodies: used by immune system to help identify foreign material or specific antigens in the blood - MHC proteins: mark cells as belonging to ‘self’ - glycoproteins: are cell markers that are a combination of protein and carbohydrate. Membrane proteins - act as channels for specific molecules to cross membranes. 1 - act as carriers for specific molecules to cross membranes. - act as receptors for very specific molecules (ex. there are receptor proteins for insulin) which can signal certain cellular functions. y - act structurally to shape cells og enzyme proteins - cytochrome c: involved in glucose metabolism. - lactase: breaks lactose into it’s monosaccharides ol - pepsin: breaks protein down into small peptides in the stomach. - trypsin: breaks specific peptide bonds between amino acids in the small intestine. Bi B. Nucleic acids DNA and RNA are nucleic acids – molecules of heredity. al They are macromolecules made up of a large number of nucleotides. A nucleotide is composed of a base, a sugar, and a phosphate group. er A nucleoside is composed of a base and a sugar (no phosphate). The sugar in DNA is deoxyribose, the sugar in RNA is ribose. en Nucleotide: deoxyadenosine 5’-triphosphate (dATP) -G U D Ad A nucleoside is a nucleotide without the phosphate  DNA and RNA bases: Pyrimidines 1 y og ol Purines: Bi al er en The nucleosides: (deoxy)cytidine, (deoxy)thymidine, (deoxy)uridine, (deoxy)adenosine, -G (deoxy)guanosine The nucleotides: deoxyadenosine 5’-triphosphate (dATP); adenosine monophosphate (AMP) etc. DNAis a long chain of deoxyribose units linked by phosphodiester links. The phosphate on the 5’ carbon is linked to the –OH on the 3’ carbon along the chain. U On each deoxyribose there is a base. D Ad  The chain has two ends. The 5’ end and the 3’ end. It is not symmetrical. The primary sequence is the linear sequence of the bases. By convention, the nucleotide sequence is specified in the 5’ to 3’ direction. The secondary structure of DNA is a right-handed double helix. The two chains in the helix run in opposite directions. The deoxyribose and phosphate groups run along the outside of the helix, with the negative charges outside. The bases point inwards and the flat planes are perpendicular to the helix. The two chains are held together by hydrogen bonds between the bases. The two strands are complementary in their sequence due to the specificity of base-pairing. 1 Adenine always pairs with Thymine; Guanine always pairs with Cytosine. y og ol Bi al er Melting and reannealing en High temperature and/or low salt concentration causes the two strands to melt or disassociate. If you then lower the temperature or increase the salt concentration, the two melted strands -G will re-anneal into a double helix. Hybridisation: in a mixture of DNA with different sequences, the complementary strands will find each other in the mixture. The E.coli genome U E.coli has 4.7 x 106 base pairs in a single circular double-stranded molecule. The length of the E.coli DNA is 1.4 mm. D The DNA in E.coli is tightly packaged – the bacterium is only 3 cm long. Ad The human genome The human genome (diploid) consists of about 6 x 109 base pairs of DNA. The DNA is divided into chromosomes that each contain a linear double-helical DNA molecule of about 200 x 106 base pairs. Prior to cell division, the DNA condenses into discrete chromosomes, visible by microscopy. A diploid cell has 46 chromosomes; 22 pairs of ‘normal’ chromosomes and 2 sex chromosomes. Enzymes I. Enzymes and Life Processes  The living cell is the site of tremendous biochemical activity called metabolism. This is the process of chemical and physical change which goes on continually in the living organism. Build-up of new tissue, replacement of old tissue, conversion of food to energy, disposal of waste materials, reproduction - all the activities that we characterize as "life." This building up and tearing down takes place in the face of an apparent paradox. The greatest majority of these biochemical reactions do not take place spontaneously. The phenomenon of catalysis makes possible biochemical reactions necessary for all life processes. Catalysis is defined as the acceleration of a chemical reaction by some substance which itself undergoes no permanent chemical change. The catalysts of biochemical reactions are enzymes and are responsible for bringing about almost all of the chemical reactions in living organisms. 1 Without enzymes, these reactions take place at a rate far too slow for the pace of metabolism. The oxidation of a fatty acid to carbon dioxide and water is not a gentle process in a test tube - extremes of pH, high temperatures and corrosive chemicals are required. Yet in the body, y such a reaction takes place smoothly and rapidly within a narrow range of pH and temperature. og In the laboratory, the average protein must be boiled for about 24 hours in a 20% HCl solution to achieve a complete breakdown. In the body, the breakdown takes place in four hours or less under conditions of mild physiological temperature and pH. ol Chemical Nature of Enzymes Bi All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. See Figure 1. al er en -G Basic Enzyme Reactions Enzymes are catalysts and increase the speed of a chemical reaction without U themselves undergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products. The basic enzymatic reaction can be D represented as follows Ad where E represents the enzyme catalyzing the reaction, S the substrate, the substance being changed, and P the product of the reaction. The Enzyme Substrate Complex  A theory to explain the catalytic action of enzymes was proposed by the Swedish chemist Savante Arrhenius in 1888. He proposed that the substrate and enzyme formed some intermediate substance which is known as the enzyme substrate complex. The reaction can be represented as: If this reaction is combined with the original reaction equation , the following results: 1 y og The existence of an intermediate enzyme-substrate complex has been demonstrated in the laboratory, for example, using catalase and a hydrogen peroxide derivative. At Yale ol University, Kurt G. Stern observed spectral shifts in catalase as the reaction it catalyzed proceeded. This experimental evidence indicates that the enzyme first unites in some way with Bi the substrate and then returns to its original form after the reaction is concluded. Factors Affecting Enzyme Activity al Knowledge of basic enzyme kinetic theory is important in enzyme analysis in order both to understand the basic enzymatic mechanism and to select a method for enzyme er analysis. The conditions selected to measure the activity of an enzyme would not be the same as those selected to measure the concentration of its substrate. Several factors affect the rate at which enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate en concentration, and the presence of any inhibitors or activators. Enzyme Concentration -G 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 U can be represented as: D Ad  1 y og These reactions are said to be "zero order" because the rates are independent of ol substrate concentration, and are equal to some constant k. The formation of products proceeds at a rate which is linear with time. The addition of more substrate does not serve to increase the Bi rate. In zero order kinetics, allowing the assay to run for double time results in double the amount of product. al er en -G U 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 D as temperature, pH, etc. 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 Ad limiting factor. It is satisfied only when the reaction is zero order. In Figure 5, activity is directly proportional to concentration in the area AB, but not in BC. Enzyme activity is generally greatest when substrate concentration is unlimiting.  1 y og ol Bi When the concentration of the product of an enzymatic reaction is plotted against time, a similar curve results, Figure 6. al er en -G U D Ad 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.  Figure 7 illustrates three types of reactions which might be encountered in enzyme assays and shows the problems which might be encountered if only single measurements are made. B is a straight line representing a zero order reaction which permits accurate determination of enzyme activity for part or all of the reaction time. A represents the type of reaction that was shown in Figure 6. This reaction is zero order initially and then slows, presumably due to substrate exhaustion or product inhibition. This type of reaction is sometimes referred to as a "leading" reaction. True "potential" activity is represented by the dotted line. Curve C represents a reaction with an initial "lag" phase. Again the dotted line represents the 1 potentially measurable activity. Multiple determinations of product concentration enable each curve to be plotted and true activity determined. A single end point determination at E would lead to the false conclusion that all three samples had identical enzyme concentration. y og Substrate Concentration It has been shown experimentally that if the amount of the enzyme is kept ol 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 8. Bi al er en -G U D It is theorized that when this maximum velocity had been reached, all of the Ad 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 8. 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 1 expression for the reaction velocity in terms of this constant and the substrate concentration. y og ol Bi Michaelis constants have been determined for many of the commonly used enzymes. al The size of Km tells us several things about a particular enzyme. er 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 en velocity. 3. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be the enzyme's natural substrate, though this is not true for all enzymes. -G Effects of Inhibitors on Enzyme Activity Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition. U Most theories concerning inhibition mechanisms are based on the existence of D the enzyme substrate complex ES. As mentioned earlier, the existence of temporary ES structures has been verified in the laboratory. Ad 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.  1 y og ol 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. Bi The substrate is held in such a way that its conversion to the reaction products is more favorable. If we consider the enzyme as the lock and the substrate the key (Figure 9) - 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 al 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 er 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. en 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 10. -G U D Ad  Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 11 shows the reaction velocity decreasing after the maximum velocity has been reached. 1 y og Additional amounts of substrate added to the reaction mixture after this point ol actually decrease the reaction rate. This is thought to be due to the fact that there are so many substrate molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 12) and prevent any other substrate molecules from occupying them. Bi al er en -G This causes the reaction rate to drop since all of the enzyme present is not being used. Temperature Effects Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases U as the temperature is raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 D degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. Ad As shown in Figure 13, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40·C, most enzyme determinations are carried out somewhat below that temperature. Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5·C or below is generally the most suitable. Some enzymes lose their activity when frozen.  1 y Effects of pH og Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH. This is graphically illustrated in Figure 14. ol Bi al er en Extremely high or low pH values generally result in complete loss of activity for -G 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. The optimum pH value will vary greatly from one enzyme to another, as Table II shows: U D Ad  In addition to temperature and pH there are other factors, such as ionic strength, which can affect the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimized in order for an enzymatic reaction to be accurate and reproducible. 1 y og ol Bi al er en -G U D Ad