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11/18/2014 Enzymes Hamad Yaseen, PhD Biochemistry 210 [email protected] MLS Dept, FAHS, HSC, KU Read Chapter 23 Enzymes as Biological Catalysts • Enzymes are proteins that increase the rate of reaction by lowering the energy of activation. Without themselves undergoing any changes. • They catalyze nearly all the chemical reactions taking place in the cells of the body • Enzymes have unique three-dimensional shapes that fit the shapes of reactants (substrates) 1 11/18/2014 Rate Enhancement Formation of carbonic acid from CO2 and H2O. Uncatalyzed rate = 1.3 x 10-1 molecules/sec Catalyzed rate = 1.0 x 106 molecules/sec Rate enhancement = 7.7 x 106 times Carbonic Anhydrase Table of Rate Enhancements 2 11/18/2014 Enzymes • The vast majority of enzymes are proteins. • However, proteins are not the only biological catalysts. • Ribozymes, are enzymes made of ribonucleic acid. Naming Enzymes • The name of an enzyme identifies the reacting substance - usually ends in –ase • For example, sucrase catalyzes the hydrolysis of sucrose • The name also describes the function of the enzyme • For example, oxidases catalyze oxidation reactions • Sometimes common names are used, particularly for the digestion enzymes such as pepsin and trypsin • Some names describe both the substrate and the function • For example, alcohol dehydrogenase oxides ethanol 3 11/18/2014 Enzyme Nomenclature • active site - a region of an enzyme comprised of different amino acids where catalysis occurs (determined by the tertiary and quaternary structure of each enzyme) • substrate - the molecule being utilized and/or modified by a particular enzyme at its active site • co-factor - organic or inorganic molecules that are required by some enzymes for activity. These include Mg2+, Fe2+, Zn2+ and larger molecules termed co-enzymes like nicotinamide adenine dinucleotide (NAD+), coenzyme A, and many vitamins. • prosthetic group - a metal or other co-enzyme covalently bound to an enzyme • holoenzyme - a complete, catalytically active enzyme including all co-factors • apoenzyme - the protein portion of a holoenzyme minus the co-factors • isozyme - (or iso-enzyme) an enzyme that performs the same or similar function of another enzyme. This generally arises due to similar but different genes encoding these enzymes and frequently is tissue-type specific or dependent on the growth or developmental status of an organism. Enzyme structure • Enzymes are proteins • They have a globular shape • A complex 3-D structure • Human pancreatic amylase © 2007 Paul Billiet ODWS 4 11/18/2014 The active site • One part of an enzyme, the active site, is particularly important • The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily © H.PELLETIER, M.R.SAWAYA ProNuC Database © 2007 Paul Billiet ODWS Cofactors • An additional non-protein molecule that is needed by some enzymes to help the reaction • Tightly bound cofactors are called prosthetic groups • Cofactors that are bound and released easily are called coenzymes • Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors Jmol from a RCSB PDB file © 2007 Steve Cook H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS © 2007 Paul Billiet ODWS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997) 5 11/18/2014 Classification of Enzymes • Enzymes are classified according to the type of reaction they catalyze: Class Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases Reactions catalyzed Oxidation-reduction Transfer groups of atoms Hydrolysis Add atoms/remove atoms to/from a double bond Rearrange atoms Use ATP to combine molecules Oxidoreductases, Transferases and Hydrolases 6 11/18/2014 Lyases, Isomerases and Ligases Active Site of an Enzyme • The active site is a region within an enzyme that fits the shape of substrate molecules • Amino acid side-chains align to bind the substrate through H-bonding, saltbridges, hydrophobic interactions, etc. • Products are released when the reaction is complete (they no longer fit well in the active site) 7 11/18/2014 Enzyme Specificity • Enzymes have varying degrees of specificity for substrates • Enzymes may recognize and catalyze: - a single substrate - a group of similar substrates - a particular type of bond Factors influence enzyme activity? • Enzyme and substrate concentration • Temperature • P.H 8 11/18/2014 Enzyme Concentration and Reaction Rate • The rate of reaction increases as enzyme concentration increases (at constant substrate concentration) • At higher enzyme concentrations, more enzymes are available to catalyze the reaction (more reactions at once) • There is a linear relationship between reaction rate and enzyme concentration (at constant substrate concentration) Substrate Concentration and Reaction Rate • The rate of reaction increases as substrate concentration increases (at constant enzyme concentration) • Maximum activity occurs when the enzyme is saturated (when all enzymes are binding substrate) • The relationship between reaction rate and substrate concentration is exponential, and asymptotes (levels off) when the enzyme is saturated 9 11/18/2014 Temperature and Enzyme Activity • Enzymes are most active at an optimum temperature (usually 37°C in humans) • They show little activity at low temperatures • Activity is lost at high temperatures as denaturation occurs pH and Enzyme Activity • Enzymes are most active at optimum pH • Amino acids with acidic or basic side-chains have the proper charges when the pH is optimum • Activity is lost at low or high pH as tertiary structure is disrupted 10 11/18/2014 Optimum pH for Selected Enzymes • Most enzymes of the body have an optimum pH of about 7.4 • However, in certain organs, enzymes operate at lower and higher optimum pH values What are the mechanisms of enzyme action? • Lock-and-key model • Induced fit model • Catalytic power of enzymes 11 11/18/2014 Lock-and-Key Model • In the lock-and-key model of enzyme action: - the active site has a rigid shape - only substrates with the matching shape can fit - the substrate is a key that fits the lock of the active site • This is an older model, however, and does not work for all enzymes Lock & Key – Fischer (1894) A proposal for ES 12 11/18/2014 Induced Fit Model • In the induced-fit model of enzyme action: - the active site is flexible, not rigid - the shapes of the enzyme, active site, and substrate adjust to maximumize the fit, which improves catalysis - there is a greater range of substrate specificity • This model is more consistent with a wider range of enzymes Induced Fit – Koshland (1963) A proposal for ES 13 11/18/2014 Catalytic power of enzymes • Previous models emphasized on the shape of the active site. However the chemistry at the active site is actually the most important factor. Enzyme Catalyzed Reactions • When a substrate (S) fits properly in an active site, an enzyme-substrate (ES) complex is formed: E + S ES • Within the active site of the ES complex, the reaction occurs to convert substrate to product (P): ES E + P • The products are then released, allowing another substrate molecule to bind the enzyme - this cycle can be repeated millions (or even more) times per minute • The overall reaction for the conversion of substrate to product can be written as follows: E + S ES E + P 14 11/18/2014 Example of an Enzyme Catalyzed Reaction • The reaction for the sucrase catalyzed hydrolysis of sucrose to glucose and fructose can be written as follows: E + S ES E + P1 + P2 where E = sucrase, S = sucrose, P1 = glucose and P2 = fructose Isoenzymes • Isoenzymes are different forms of an enzyme that catalyze the same reaction in different tissues in the body - they have slight variations in the amino acid sequences of the subunits of their quaternary structure • For example, lactate dehydrogenase (LDH), which converts lactate to pyruvate, consists of five isoenzymes 15 11/18/2014 How are Enzymes Regulated? • Feedback Control: • An enzyme regulation process in which formation of a product inhibits an earlier reaction in the sequence. • The reaction product of one enzyme may control the activity of another, especially in a complex system in which enzymes work cooperatively. How are Enzymes Regulated? • Proenzymes • Some enzymes are manufactured in the body in an inactive form. To make them active, a small part of their polypeptide chain must be removed. • Why this is important? Think trypsin 16 11/18/2014 How are Enzymes Regulated? • Allosterism • An event that occurs at a site other than the active site but that eventually affects the active site. • Any enzyme regulated by this mechanism is called Allosteric enzyme. • These could be: Negative modulators and positive modulators Enzyme Inhibitors • Inhibitors (I) are molecules that cause a loss of enzyme activity • They prevent substrates from fitting into the active site of the enzyme: E + S ES E + P E + I EI no P formed 17 11/18/2014 Reversible Inhibitors (Competitive Inhibition) • A reversible inhibitor goes on and off, allowing the enzyme to regain activity when the inhibitor leaves • A competitive inhibitor is reversible and has a structure like the substrate - it competes with the substrate for the active site - its effect is reversed by increasing substrate concentration Reversible Inhibitors (Noncompetitive Inhibition) • A noncompetitive inhibitor has a structure that is different than that of the substrate - it binds to an allosteric site rather than to the active site - it distorts the shape of the enzyme, which alters the shape of the active site and prevents the binding of the substrate • The effect can not be reversed by adding more substrate 18 11/18/2014 How are Enzymes Regulated? • Protein Modification • The modification is usually change in primary structure, typically by addition of a functional group covalently bound to the apoenzyme. • A well described example: Phosphorylation How are Enzymes Regulated? • Isoenzymes • Regulatory of enzyme activity via having more than one form of the enzymes in different tissues and organs. A good example is Lactate dehydrogenase (LDH). 19 11/18/2014 Isoenzymes • Why this is important? “The heart example” • The heart is purely aerobic organ. • LDH is used to convert lactate into pyruvate. • The H4 enzyme is allosterically inhibited by high levels of pyruvate (its product). • Has a higher affinity for lactate (its substrate) than M4 enzyme, which is optimized for the opposite reaction. The M4 isoenzyme favors the production of lactate. Diagnostic Enzymes • The levels of diagnostic enzymes in the blood can be used to determine the amount of damage in specific tissues 20