Enzymes PDF

Enzymes Dr. Margaret Doherty  What is an enzyme? A type of protein that speeds up a chemical reaction They have a globular shape They can have a complex tertiary (3D) structure e.g. Enzymes in your saliva (amylase) speed up digestion by breaking down starc...

Enzymes Dr. Margaret Doherty  What is an enzyme? A type of protein that speeds up a chemical reaction They have a globular shape They can have a complex tertiary (3D) structure e.g. Enzymes in your saliva (amylase) speed up digestion by breaking down starches into sugar They can be damaged/destroyed by protein denaturing materials e.g. heating, pH, and certain denaturing chemicals Factors That Influence Enzyme Activity Temperature pH Substrate Concentration Enzyme Concentration Cofactors & Coenzymes Inhibitors  Principles of Catalysis A catalyst is a substance that increases the rate of a reaction without itself being consumed by the process A reaction in which a catalyst is involved is called a catalyzed reaction and the process is called catalysis A catalyst lowers the Gibbs energy of activation by providing a different mechanism for the reaction  How enzymes work A chemical reaction A to B goes through a transition state This state has a greater amount of energy than either A or B Therefore, the molecules must have sufficient energy to form the transition state otherwise, they will not react The rate of the forward reaction depends on the temperature and on the difference in free energy between that of A and the transition state This energy is called the Gibbs free energy of activation or the activation barrier (DG) Enzyme Action  Substrate The reacting molecule that binds to the enzyme is called the substrate Enzymes are specific to their substrates The specificity is determined by the active site  Specificity Some enzymes act on many compounds having a common structural feature e.g. phosphatase Relatively broad specificity Enzymes are usually very specific e.g. aspartase deaminates (i.e. removes an amino group) aspartic acid in a reversible reaction to produce fumaric acid Aspartase has rigid stereo and geometrical specificity It is stereospecific since it will not deaminate D-aspartic acid Geometrically specific will not add ammonia to maleic acid, a cis-isomer of fumaric acid  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  Active site Reaction occurs in the active site Relatively small part of the enzyme visualized as a cleft in the surface of the molecule R-groups of key amino acid residues in the active site Bind the substrates to the site Catalyse forming product molecules  Substrate binding to the active site Substrate must possess certain groups Require a binding group binds to the enzyme and positions the substrate molecule Ensure bond on the substrate is properly located in relation to the catalytic site of the enzyme The induced-fit theory As a substrate approaches the active site Both the substrate and the enzyme change enables the substrate to be bound to the active site Only upon approach/binding that the active site has a shape complementary to that of the substrate This process of dynamic recognition is called induced fit Induced Fit Theory  Kinetics Is the study of the rates(velocities) of chemical reactions Measures changes in the concentration of substrate and/or products of a reaction over time to determine the velocity of the reaction Measures the effects of concentration, temperature, pH etc. to characterize the properties of the enzyme catalyzing the reaction The Equations of Enzyme Kinetics In enzyme kinetics, it is customary to measure the initial rate (v0) of a reaction to minimize reversible reactions and the inhibition of enzymes by products Plot of the initial rate of an enzyme-catalyzed reaction versus substrate concentration First Order Reaction Kinetics First Order Reaction Kinetics Rate is directionally proportional to [A] Enzyme catalysed reaction  Formation and fate of ES complex Binding involves the formation of a covalent intermediate enzyme-substrate complex (ES) Formation is the first step in enzymatic catalysis Two possible fates: It can dissociate to free enzyme (E) and free substrate (S) Alternatively, it can proceed to form product, P by forming an enzyme-product complex (EP) which then dissociates to form the free enzyme (E) and product (P) molecules  Michaelis-Menten k2 E+S↔ ES ↔ k1 k k E + P -1 -2 E is the enzyme, S is the substrate, ES is the enzyme-substrate complex, and P is the product. E combines with the S in order to form the ES complex, which in turn converts to P while preserving the enzyme. Michaelis-Menten Kinetic Parameters Lineweaver-Burk Plot Effect of Temperature on Enzyme Activity Effect of pH on Enzyme Activity Optimum pH values Enzyme activity Trypsin Pepsin 1 3 5 7 9 11 pH Effect of Substrate on Enzyme Activity Effect of Enzyme Concentration on Enzyme Activity  Cofactor 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 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)  Coenzymes Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Coenzymes transport chemical groups from one enzyme to another Enzyme along with a co-enzyme is known as - holoenzyme Enzyme without a co-enzyme is known as - apoenzyme Enzyme Inhibition Alteration in the enzyme activity by specific substances other than non-specific substances e.g. pH, temperature etc. 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.  The effect of enzyme inhibition 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.  The effect of enzyme inhibition Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories. Competitive Non-Competitive  The effect of enzyme inhibition Competitive Enzyme Inhibitors Prevent the formation of Enzyme-Substrate Complexes because they have a similar shape to the substrate molecule. This means that they fit into the Active Site, but remain unreacted since they have a different structure to the substrate. Usually temporary. The level of inhibition depends on the relative concentrations of substrate and Inhibitor, since they are competing for places in enzyme active sites.  The effect of enzyme inhibition Non-competitive Enzyme Inhibitors work by preventing the formation of Enzyme-Product Complexes. Usually, Non-competitive Inhibitors bind to a site other than the Active Site, called an Allosteric Site. Doing so distorts the tertiary structure of the enzyme, such that it can no longer catalyse a reaction. Enzyme Inhibition Competitive and Non-Competitive Enzyme Inhibition Impact on Health Toxic effects Organophosphorus (nerve gas) Mercury containing compounds Cyanide (irreversible Inhibitor of the enzyme Cytochrome C Oxidase) Carbon monoxide Hydrogen sulphide Irreversible inhibition by covalent binding Irreversible inhibition of essential enzymes Results in high toxicity Applications of inhibitors Sulpha drugs Methotrexate Anticholinesterases Allopuriinol Disulfiram NRTIs Protease inhibitors Viagra SAIDs NSAIDs Orlistat Trimethoprim  Conclusion Enzymes are organic catalysts The reaction(s) which is catalysed by a particular enzyme depends on the structure of the enzyme and, in particular, the active site of the enzyme Some enzymes are very specific in that they can discriminate between closely related compounds The ability of enzymes to accelerate reactions depends on: Availability of appropriate cofactors The energy required to form the transition state complex Conclusion Michaelis–Menten enzyme kinetics describes the behavior of enzymes over a wide range of substrate concentrations. Two important kinetic parameters are Vmax and KM. Vmax is the maximal velocity of an enzyme-catalyzed reaction at saturating substrate concentration. KM is loosely related to substrate affinity, but a more correct description is that it determines the shape of the Michaelis– Menten hyperbolic curve. Conclusion Kinetic experiments can quantify how the rate of an enzyme- catalyzed reaction changes with the concentration of substrate. Data obtained can be plotted and fit to the Michaelis–Menten equation or rearranged into a double-reciprocal plot. From these graphs, the kinetic parameters KM and Vmax can be obtained