1. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini This document is licensed under the Attribution-NonCommercial-ShareAlike 2.5 Italy license, available athttp://creativecommons.org/licenses/by-nc-sa/2.5/it/ ENZYMES AND METABOLIC PATHWAYS

2. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 1. Chemical reactions in cells Thousands of biochemical reactions, in which metabolites are converted into each other and macromolecules are build up, proceed at any given instant within living cells. However, the greatest majority of these reactions would occour spontaneously at extremely low rates. Thousands of biochemical reactions, in which metabolites are converted into each other and macromolecules are build up, proceed at any given instant within living cells. However, the greatest majority of these reactions would occour spontaneously at extremely low rates.  For example, the oxidation of a fatty acid to carbon dioxide and water in a test tube requires extremes of pH, high temperatures and corrosive chemicals. Yet in the cell, such a reaction takes place smoothly and rapidly within a narrow range of pH and temperature. As another example, 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. How can living things perform the magic of speeding up chemical reactions many orders of magnitude, specifically those reactions they most need at any given moment? How can living things perform the magic of speeding up chemical reactions many orders of magnitude, specifically those reactions they most need at any given moment?

3. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 2. Introducing enzymes The ENZYMES are the driving force behind all biochemical reactions happening in cells. The ENZYMES are the driving force behind all biochemical reactions happening in cells. Enzymes lower the energy barrier between reactants and products, thus increasing the rate of the reaction. Enzymes lower the energy barrier between reactants and products, thus increasing the rate of the reaction. Enzymes are biological catalysts. A catalyst is a species that accelerates the rate of a chemical reaction whilst remaining unchanged at the end of the reaction. Catalysis is achieved by reducing the activation energy for the reaction. Enzymes are biological catalysts. A catalyst is a species that accelerates the rate of a chemical reaction whilst remaining unchanged at the end of the reaction. Catalysis is achieved by reducing the activation energy for the reaction. Enzymes can catalyse reactions at rates typically 10 6 to 10 14 times faster than the uncatalysed reaction. Enzymes can catalyse reactions at rates typically 10 6 to 10 14 times faster than the uncatalysed reaction. Enzymes are very selective about substrates they act upon and also where the chemistry takes place on a substrate. Enzymes are very selective about substrates they act upon and also where the chemistry takes place on a substrate. Both the forward and reverse reactions are catalysed. A catalyst cannot change the position of thermodynamic equilibrium, only the rate at which it is attained. Both the forward and reverse reactions are catalysed. A catalyst cannot change the position of thermodynamic equilibrium, only the rate at which it is attained.

4. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 3. Enzymes are proteins Enzymes are composed of proteins, and proteins are long polymers of amino acids. Amino acids all have this general formula: Enzymes are composed of proteins, and proteins are long polymers of amino acids. Amino acids all have this general formula: Amino acids have two functional groups (aminic and carbossilyc), which can react together forming covalent bonds called peptide bonds, so that they are linked head-to-tail. Amino acids have two functional groups (aminic and carbossilyc), which can react together forming covalent bonds called peptide bonds, so that they are linked head-to-tail. The side chain, or R group, can be anything from a hydrogen atom (as in the amino acid glycine) to a complex ring (as in the amino acid tryptophan). The side chain, or R group, can be anything from a hydrogen atom (as in the amino acid glycine) to a complex ring (as in the amino acid tryptophan). Each of the 20 amino acids known to occur in proteins has a different R group that gives it its unique properties. Each of the 20 amino acids known to occur in proteins has a different R group that gives it its unique properties. The linear sequence of the amino acids in a polypeptide chain constitutes the primary structure of the protein The linear sequence of the amino acids in a polypeptide chain constitutes the primary structure of the protein

5. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 4. Four levels of structure of the proteins Proteins have a complex structure that is traditionally thought of as having four levels.  The primary structure of a protein is the sequence of amino acids in its polypeptide chain.  The secondary structure is the regular arrangement of amino acids within localized regions of the polypeptide.  The tertiary structure is the folding of the polypeptide chain as a result of interactions between the side chains of amino acids.  The fourth level of protein structure, quaternary structure, consists of the interactions between different polypeptide chains in proteins composed of more than one polypeptide. Many proteins are compact structures; such proteins are called globular proteins. Enzymes and antibodies are among the important globular proteins. Other, unfolded proteins, called fibrous proteins, are important components of such structures as hair and muscle.

6. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 2. The sequence of aminoacids The peptide bond. (a) A polypeptide is formed by the removal of water between amino acids to form peptide bonds. Each aa indicates an amino acid. R1, R2, and R3 represent R groups (side chains) that differentiate the amino acids. R can be anything from a hydrogen atom (as in glycine) to a complex ring (as in tryptophan). (b) The peptide group is a rigid planar unit with the R groups projecting out from the CN backbone. Standard bond distances (in angstroms) are shown.

7. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 5. Primary structure Linear sequences of two proteins. (a) The E. coli tryptophan synthetase A protein, 268 amino acids long. (b) Bovine insulin protein. Note that the amino acid cysteine can form unique “sulfur bridges,” because it contains sulfur.

8. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 6. Secondary structures The secondary structure of a protein refers to the interrelations of amino acids that are close together in the linear sequence. Polypeptides can bend into regularly repeating (periodic) structures, created by hydrogen bonds between the CO and NH groups of different residues. Two of the basic periodic structures are the α helix and the β pleated sheet. Two views of the antiparallel β pleated sheet, another common form of secondary protein structure. Adjacent strands run in opposite directions. Hydrogen bonds between NH and CO groups of adjacent strands stabilize the structure. The side chains (R) are above and below the plane of the sheet. The α helix, a common basis of secondary protein structure. Each R is a specific side chain on one amino acid. The black dots represent weak hydrogen bonds that bond the CO group of residue n to the NH group of residue n + 4, thereby stabilizing the helical shape.

9. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 7. Terziary structure A protein also has a three-dimensional architecture, termed the tertiary structure, which is created by electrostatic, hydrogen, and Van der Waals bonds that form between the various amino acid R groups, causing the protein chain to fold back on itself. In many cases, amino acids that are far apart in the linear sequence are brought close together in the tertiary structure. A protein also has a three-dimensional architecture, termed the tertiary structure, which is created by electrostatic, hydrogen, and Van der Waals bonds that form between the various amino acid R groups, causing the protein chain to fold back on itself. In many cases, amino acids that are far apart in the linear sequence are brought close together in the tertiary structure. Folded tertiary structure of myoglobin, an oxygen-storage protein. Each dot represents an amino acid. The heme group, a cofactor that facilitates the binding of oxygen, is shown in blue.

10. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 8. The four levels of protein structure Levels of protein structure. Levels of protein structure.  Primary structure.  Secondary structure. The polypeptide shown in part a is drawn into an α helix by hydrogen bonds.  Tertiary structure: the three-dimensional structure of myoglobin.  Quaternary structure: the arrangement of two α subunits and two β subunits to form the complete quaternary structure of hemoglobin.

11. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 9. The active site Enzymes are typically large proteins, which are structured specifically for the reaction they catalyze. Their size provide sites for action and stability of the overall structure. Enzymes are typically large proteins, which are structured specifically for the reaction they catalyze. Their size provide sites for action and stability of the overall structure. Two important sites within enzymes are: Two important sites within enzymes are:  The catalytic site, which is a region within the enzyme involved with catalysis, and  The substrate binding site which is the specific area on the enzyme to which reactants called substrates bind to. The catalytic site and substrate binding site are often close or overlapping and collectively they are called the active site. The catalytic site and substrate binding site are often close or overlapping and collectively they are called the active site.  If the catalytic site is not near the substrate binding site it can move into position once the enzyme is bound to a substrate.

12. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 10. The “Lock-and-key” metaphor Schematic representation of the action of a hypothetical enzyme in putting two substrate molecules together. (a) In the "lock-and-key" mechanism the substrates have a complementary fit to the enzyme's active site. (b) In the induced-fit model, binding of substrates induces a conformational change in the enzyme.

13. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 11. Aditional components of enzymes Often enzymes require additional components to become active. These may be: Often enzymes require additional components to become active. These may be:  co-factors: simple cations, or small organic or inorganic molecules that bind loosely to the enzyme,  prosthetic groups: similar to co-factors but more tightly bound to the enzyme, or  co-enzymes – which are more complex than co-factors and prosthetic groups, they often act as a second substrate or bind covalently with the enzyme to affect the active site.

14. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 12. The first step of photosynthesis Photosynthesis. The key passage of the photosynthesis is the organication of the carbon, or the fixation of CO 2. The CO 2 molecule condenses with ribulose 1,5-bisphosphate to form an unstable six-carbon compound, which is rapidly hydrolyzed to two molecules of 3-phosphoglycerate. This reaction is catalyzed by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO)

15. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 13. An enzyme of fundamental importance for life The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is located on the stromal surface of the thylakoid membranes of chloroplasts. It comprises eight large (L) subunits (one shown in red and the others in yellow) and eight small (S) subunits (one shown here in blue and the others in white). The active sites lie in the L subunits. Each L subunit contains a catalytic site and a regulatory site. The S chains enhance the catalytic activity of the L chains. This enzyme is very abundant, constituting more than 16% of chloroplast total protein. RUBISCO is probably the most abundant protein in the biosphere.

16. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 14. The active site of RUBISCO Structure of the catalytic domain of the active form of ribulose 1,5-bisphosphate carboxylase. Dark blue cylinders represent  helices and yellow arrows represent  sheets in the polypeptide. The key residues in the active site are carbamylated lysine 191, aspartate 193, and glutamate 194; a Mg 2+ ion is bound to carbamylated lysine 191. The substrates CO 2 and ribulose 1,5-bisphosphate are shown bound to the active site.

17. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 15. METABOLIC PATHWAYS There are thousands of enzyme-catalyzed reactions in a cell. If the biochemical reactions involved in this process were reversible, we would convert our macromolecules back to metabolites if we stop eating even for a short period of time. There are thousands of enzyme-catalyzed reactions in a cell. If the biochemical reactions involved in this process were reversible, we would convert our macromolecules back to metabolites if we stop eating even for a short period of time. To prevent this from happening, our metabolism is organized in metabolic pathways. These pathways are a series of biochemical reactions which are, as a whole, irreversible. To prevent this from happening, our metabolism is organized in metabolic pathways. These pathways are a series of biochemical reactions which are, as a whole, irreversible. These reactions are organized in consecutive steps or pathways where the products of one reaction can become the reactants in another. Every biochemical molecule is synthesized in a biochemical pathway with specific enzymes. These reactions are organized in consecutive steps or pathways where the products of one reaction can become the reactants in another. Every biochemical molecule is synthesized in a biochemical pathway with specific enzymes.

18. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 16. Metabolic networks A metabolic network is the complete set of metabolic and physical processes that determine the physiological and biochemical properties of a cell. As such, these networks comprise the chemical reactions of metabolism as well as the regulatory interactions that guide these reactions. With the sequencing of complete genomes, it is now possible to reconstruct the network of biochemical reactions in many organisms, from bacteria to human. Several of these networks are available online

19. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 17. Metabolic pathways of phenylalanine in human One small part of the human metabolic map, showing the consequences of various specific enzyme failures. (Disease phenotypes are shown in colored boxes.)

20. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 18. Defects in amino acid metabolism The most frequent defects in amino acid metabolism involve the amino acids phenylalanine and tyrosine. The most frequent defects in amino acid metabolism involve the amino acids phenylalanine and tyrosine. Numerous enzymes are required to convert phenylalanine into a variety of biochemical products. The metabolism of phenylalanine and the various metabolic blocks are illustrated in graphic on the left where for simplicity, many intermediate steps have been omitted. Numerous enzymes are required to convert phenylalanine into a variety of biochemical products. The metabolism of phenylalanine and the various metabolic blocks are illustrated in graphic on the left where for simplicity, many intermediate steps have been omitted. The circled letters indicate enzyme defects which will of course disrupt the reactions which follow it. The circled letters indicate enzyme defects which will of course disrupt the reactions which follow it.

21. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 19. Phenylketonuria Phenylketonuria is caused by an absence or deficiency of phenylalanine hydroxylase or, more rarely, of its tetrahydrobiopterin cofactor. Phenylalanine accumulates in all body fluids because it cannot be converted into tyrosine. Normally, three-quarters of the phenylalanine is converted into tyrosine, and the other quarter becomes incorporated into proteins. The accumulation of phenylpyruvate leads to severe mental retardation in infants. If the high level of phenylpyruvic acid is detected soon after birth, the baby can be placed on a special low-phenylalanine diet and develops without retardation. Because the major outflow pathway is blocked in phenylketonuria, the blood level of phenylalanine is typically at least 20-fold as high as in normal people. Minor fates of phenylalanine in normal people, such as the formation of phenylpyruvate, become major fates in phenylketonurics.

22. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 20. Goitrous cretinism and Albinism Goitrous Cretinism Hypothyroidism results from the absence of an enzyme to incorporate iodine into tyrosine in the first step in the synthesis of thyroxine. The result is stunted growth, lethargy, course hair, poor muscle tone and other facial defects. Hypothyroidism is treated by administration of thyroid extract. Albinism The biochemical defect in albinism appears to be the absence of the enzyme tyrosinase, which prevents the synthesis of melanin pigment by pigment-forming cells. These individuals have a very white skin, fine white hair, pink or light blue irises of the eyes, and a variety of other eye disturbances. Various types of localized albinism are characterized by the absence of pigment in specific parts of the body. There is no treatment for albinism.

23. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini 21. Tyrosinosis and Alkaptonuria Tyrosinosis Accumulation of p-hydroxyphenylpyruvic acid usually leads to an enlargement of the liver and spleen. Death results from liver failure between 4 months and 5 years of age. Diet control may help in reducing the symptoms of tyrosinosis. Alkaptonuria Alkaptonuria occurs when the absence of an enzyme prevents the breakdown of homogentisic acid. A large amount of homogentisic acid excreted in the urine causes it to turn black upon exposure to air. Other characteristics of alkaptonuria include arthritis and pigmentation of cartilage. It was this relatively benign disease that was first used as the basis for the concept of inborn errors of metabolism.