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Enzymes Enzymes are PROTEINS and certain class of RNA (ribozymes) which ENHANCE the rate of THERMODYNAMICALLY feasible REACTION and are NOT PERMANENTLY ALTERED IN THE PROCESS Chemical reactions Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY During this part of the reaction the molecules are said to be in a transition state. Reaction pathway © 2007 Paul Billiet ODWS Making reactions go faster Temperature increase molecules movement Biological systems are very sensitive to temperature changes Enzymes can increase the rate of reactions without increasing the temperature They do this by lowering the activation energy They create a new reaction pathway “a short cut” © 2007 Paul Billiet ODWS An enzyme controlled pathway Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions. © 2007 Paul Billiet ODWS The Enzyme Reaction Conventionally we say the enzyme acts on the substrate (S) to yield products (P) E S→P Since E is a catalyst it remains unchanged at the end of the reaction Each reaction has a transition state where the substrate is in an unstable, short-lived chemical/structural state. Free Energy of Activation is symbolized by ΔG‡. Enzymes act by lowering the free energy of the transition state Enzymes speed up metabolic reactions by lowering energy barriers Enzyme speed reactions by lowering EA. The transition state can be reached at moderate temperatures. Enzymes do not change delta G. It speed-up reactions that would occur eventually. The Enzyme Reaction Enzymes lower the free energy of activation by binding the transition state of the reaction better than the substrate The enzyme must bind the substrate in the CORRECT ORIENTATION otherwise there would be NO REACTION Not a lock & key but induced fit theory the enzyme and/or the substrate distort towards the transition state Biochemistry, Stryer, 5th edition The Lock and Key Hypothesis Fit between the SUBSTRATE and the ACTIVE SITE of the enzyme Same as KEY FITS INTO A LOCK very precisely © 2007 Paul Billiet ODWS The Lock and Key Hypothesis (cont.,) Temporary structure called the enzymesubstrate complex formed PRODUCTS have a different shape from the SUBSTRATE Once formed, PRODUCTS are released from the active site Leaving it free to become attached to another substrate © 2007 Paul Billiet ODWS The Lock and Key Hypothesis (cont.,) Figure 8.9. Lock-and-Key Model of Enzyme-Substrate Binding. In this model, the active site of the unbound enzyme is complementary in shape to the substrate. Biochemistry, Stryer, 5th edition The Induced Fit Hypothesis The enzyme changes shape on substrate binding The active site forms a shape complementary to the substrate only after the substrate has been bound SUBSTRATE BINDING INDUCES A CHANGE IN THE ENZYME’S CONFORMATION Induced-Fit Model of Enzyme-Substrate Binding Figure 8.10. Induced-Fit Model of Enzyme-Substrate Binding. In this model, the enzyme changes shape on substrate binding. The active site forms a shape complementary to the substrate only after the substrate has been bound. Biochemistry, Stryer, 5th edition Enzyme structure Enzymes are proteins in nature They have a globular shape A complex 3-D structure Human pancreatic amylase © 2007 Paul Billiet ODWS © Dr. Anjuman Begum The active site Is one part of the enzyme and it is particularly IMPORTANT The SHAPE and the CHEMICAL ENVIRONMENT inside the active site © H.PELLETIER, M.R.SAWAYA ProNuC Database © 2007 Paul Billiet ODWS permits a chemical reaction to proceed more easily The active site (cont.,) Typically a pocket or groove on the surface of the protein into which the substrate fits The specificity of an enzyme fit between the active site and the substrate Enzyme changes shape tighter induced fit, bringing chemical groups in position to catalyze the reaction The active site is an enzyme’s catalytic center In most cases substrates are held in the active site by WEAK INTERACTIONS A single enzyme molecule can catalyze thousands or more reactions a second Enzymes are UNAFFECTED by the reaction and are REUSABLE Enzymes can be classified according to the chemical reactions they catalyze 1. Oxidoreductases: oxidation/reduction (eg. DEHYDROGENASES) 2. Transferases: group transfer (eg. KINASES) 3. Hydrolases: hydrolysis (eg. PROTEASES) Enzymes can be classified according to the chemical reactions they catalyze 4. Lyases: lysis, generating double bond (eg. SYNTHASES) 5. Isomerases: rearrangement (eg. RACEMASES) 6. Ligases: ligation requiring ATP (eg. SYNTHETASES) Cofactors An additional non-protein molecule that is needed by some enzymes to help the reaction Nitrogenase enzyme with Fe, Mo and ADP cofactors © 2007 Paul Billiet ODWS 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 IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997) Cofactors (cont.,) Tightly bound cofactors are called PROSTHETIC GROUPS Cofactors that are bound and released easily are called COENZYMES Many vitamins are coenzymes © 2007 Paul Billiet ODWS 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 IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997) Common Coenzymes The substrate SUBSTRATES are REACTANTS activated by enzyme Enzymes are specific to their substrates The specificity is determined by the active site © 2007 Paul Billiet ODWS Substrate orientation Substrate specificity Enzymes are substrate specific When a substrate(s) binds to an enzyme, the enzyme catalyzes the conversion of the substrate to the product. – SUCRASE is an enzyme that binds to sucrose and breaks the disaccharide into fructose and glucose Enzymes are substrate specific (cont.,) The rate that a specific number of enzymes converts substrates to products depends in part on substrate concentrations At low substrate concentrations, an increase in substrate speeds binding to available active sites – However, there is a limit to how fast a reaction can occur Enzymes are substrate specific (cont.,) At some substrate concentrations, the active sites on all enzymes are engaged, called ENZYME SATURATION The only way to increase productivity at this point is to add more enzyme molecules Factors affecting Enzymes substrate concentration pH temperature inhibitors © 2007 Paul Billiet ODWS Substrate concentration: Non-enzymic reactions Reaction velocity Substrate concentration The increase in velocity is proportional to the substrate concentration © 2007 Paul Billiet ODWS Substrate concentration: Enzymic reactions Vmax Reaction velocity Substrate concentration Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. If you alter the concentration of the enzyme then Vmax will change too. © 2007 Paul Billiet ODWS The effect of pH Optimum pH values Enzyme activity Trypsin Pepsin 1 © 2007 Paul Billiet ODWS 3 5 7 pH 9 11 The effect of pH At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur Extreme pH levels will produce DENATURATION OF ENZYME The structure of the enzyme is changed The active site is distorted and the substrate molecules will no longer fit in it © 2007 Paul Billiet ODWS The effect of temperature LITTLE activity at LOW temperature Rate INCREASES with temperature Most active at optimum temperatures (usually 37°C in humans) Enzyme-controlled reactions follow this rule as they are chemical reactions BUT at high temperatures proteins DENATURE © 2007 Paul Billiet ODWS The effect of temperature Q10 Enzyme activity 0 © 2007 Paul Billiet ODWS 10 20 30 40 Temperature / °C Denaturation 50 Inhibitors Inhibitors are CHEMICALS that reduce the rate of enzymic reactions. The are usually SPECIFIC and they work at low concentrations. They BLOCK the enzyme but they do not usually destroy it. Many DRUGS and POISONS are inhibitors of enzymes in the nervous system. © 2007 Paul Billiet ODWS Types of inhibitors Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly. Examples: NERVE GASES and PESTICIDES, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase. © 2007 Paul Billiet ODWS Types of inhibitors (cont.) Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories COMPETITIVE NON-COMPETITIVE © 2007 Paul Billiet ODWS Types of inhibitors (cont.) COMPETITIVE: These compete with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Resembles the substrate’s structure closely. © 2007 Paul Billiet ODWS E+I Reversible reaction EI Enzyme inhibitor complex Types of inhibitors (cont.) NON-COMPETITIVE: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. Examples Cyanide combines with the Iron in the enzymes cytochrome oxidase. Heavy metals, Ag or Hg, combine with –SH groups. © 2007 Paul Billiet ODWS Enzymes are used in biological washing powders PROTEASES break down the coloured, insoluble proteins that cause stains to smaller, colourless soluble polypeptides. LIPASES can hydrolyze lipid spots at lower temperatures Enzymes are used in the food industry PECTINASE break down substances in apple cell walls and enable greater juice extraction. Enzymes are used in the food industry LACTASE breaks down lactose in milk into glucose and galactose. This makes milk drinkable for lactose intolerant people.