* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Enzymes
Basal metabolic rate wikipedia , lookup
Magnesium in biology wikipedia , lookup
Lipid signaling wikipedia , lookup
Metabolic network modelling wikipedia , lookup
Nicotinamide adenine dinucleotide wikipedia , lookup
Western blot wikipedia , lookup
Deoxyribozyme wikipedia , lookup
NADH:ubiquinone oxidoreductase (H+-translocating) wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Restriction enzyme wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Proteolysis wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Biochemistry wikipedia , lookup
Metalloprotein wikipedia , lookup
Biosynthesis wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Catalytic triad wikipedia , lookup
Enzymes • Catalysts speed up chemical reactions without being changed themselves • Living organisms make biological catalysts called enzymes • Enzymes are globular proteins which act as catalysts of chemical reactions • Without enzymes to catalyze them, many chemical processes happen at a very slow rate in living organisms • By making some enzymes and not others, cells can control what chemical reactions happen in their cytoplasm Introducing Enzymes • The structure of enzymes is quite delicate and can be damaged by various substances and conditions • This is called denaturation – Changing the structure of an enzyme (or other protein) so that it can no longer carry out is function • Denaturation is usually permanent in chemical reactions one or more reactants are converted into one or more products Introducing Enzymes Introducing Enzymes • In reactions catalyzed by enzymes, the reactants are called substrates substrate(s) sucrose + H2O enzyme sucrase product(s) glucose + fructose Enzyme-Substrate Specificity • Most enzymes are specific – They catalyze very few different reactions • They therefore only have a very small number of possible substrates – this is called enzyme-substrate specificity • The substrates bind to a special region on the surface of the enzyme called the active site • An active site is a region on the surface of an enzyme to which substrates bind and which catalyze a chemical reaction involving the substrates Enzyme-Substrate Specificity • The active site of an enzyme has a very intricate and precise shape • It also has distinctive chemical properties • Active sties match the shape and chemical properties of their substrates • Molecules of substrate fit the active site and are chemically attracted to it • Other molecules either do not fit or are not chemically attracted – They do not therefore bind to the active site • This is how enzymes are substrate-specific • The way in which the enzyme and substrate fit together is similar to the way in which a key fits a lock • The enzyme is like the lock and the substrate is the key that fits it Enzyme induced fit substrate enzyme • When the substrate enters the active site, the enzyme conforms (changes shape) and “hugs” the substrate • • • • • • • Substrate molecules are in continual random motion If one collides with the active site, it can bind to it The substrate fits the active site If other molecules collide with the active site they do not fit and fail to bind The active site catalyzes a chemical reaction The substrates are turned into products The products detach from the active site, leaving it free for more substrate to bind Stages in Enzyme Catalysis rate of reaction At low substrate concentrations, the enzyme activity is directly proportional to substrate concentration. This is because random collisions between substrate and active site happen more frequently with higher substrate concentrations At high substrate concentrations, all the active sites of the enzyme are fully occupied, so raising the substrate concentration has no effect concentration of substrate Effect of Substrate Concentration on Enzyme Activity rate of reaction how enzyme concentration affects rate of reaction enzyme concentration Factors Affecting Enzyme Activity • Wherever enzymes are used, it is important that they have the conditions that they need to work effectively • Temperature, pH, and substrate concentration all affect the rate at which enzymes catalyze chemical reactions • Enzymes, unlike inorganic catalysts, have optimum conditions under which they work. Effect of Temperature • Enzyme activity increases as temp increases, often doubling with every 10 oC rise • This is because collisions between substrate and active site happen more frequently at higher temps due to faster molecular motion • At high temperatures enzymes are denatured and stop working • This is because heat causes vibrations inside enzymes which break bonds needed to maintain the structure of the enzyme Effect of pH • Every enzyme has very narrow range of pH within which it works properly. • Increasing or decreasing pH – denatures the protein & slows/stops reaction rate • different enzymes have different optimal conditions for working properly Ex: temperature pH level Using Enzymes in Biotechnology • Biotechnology is the use or organisms or parts of organisms to produce things or to carry out useful processes • There are many ways in which enzymes, obtained from living organisms, can be used in biotechnology The Use of Pectinase in Fruit Juice Production • Pectin is a complex polysaccharide, found in cell walls of plants • Pectinase is an enzyme that breaks down pectin by hydrolysis reactions • Source of enzyme: – Pectinase is obtained by artificially culturing a fungus (Aspergillus niger) – The fungus grows naturally on fruits, where it uses pectinase to soften the cell walls of the fruit so that is can grow through it • Use of pectinase in biotechnology: – Fruit juices are produced by crushing ripe fruits to separate liquid juice from solid pulp – When ripe fruits are crushed, pectin form links between the cell wall and the cytoplasm of the fruit cells, making the juice viscous and more difficult to separate from the pulp – Pectinase is added during crushing of fruit to break down the pectin • Advantages: – Pectinase makes juice more fluid and easy to separate from the pulp – It therefore increases the volume of juice that is obtained – It also makes the juice less cloudy by helping solids suspended in the juice to settle be separated from the fluid Use of Protease in Biological Washing Powder • Protease enzymes break down proteins into soluble particles and amino acids • Laundry washing powders that contain protease are called biological washing powders • Source of the enzyme: – Protease is obtained by culturing a bacterium, Bacillus licheniformis, that is adapted to grow in alkaline conditions – This bacterium feeds on proteins in its habitat by secreting protease – The protease has a high pH optimum between 9 and 10 • Use of protease in biotechnology: – Detergents in laundry washing powders remove fats and oils during the washing of clothes, but much of the dirt on clothing is made of protein, not lipids – If proteases added to the washing powder, this protein is digested during the wash – The high pH optimum of the protease allows it to remain active, despite the high pH caused by alkalis in the washing powder • Advantages: – If protease is not used, protein stains on clothes can only be removed by using a very high temperature wash – Protease allows much lower temperatures to be used, with lower energy use and less risk of shrinkage of garments or loss of colored dyes Types of Enzymes • Transferases: – • Hydrolases: – • Enzymes that catalyze the removal of a groups from substrates by mechanisms other than hydrolysis Isomerases: – • enzymes that catalyze the hydrolysis of esters, cabs and proteins Lyases: – • Enzymes that catalyze the transfer of a functional group between two substrates Enzymes that catalyze the interconversion of stereoisomers and structural isomers. Ligases: – Enzymes that catalyze the linking of two compounds by breaking a phospate anhydride bond in ATP bread and cheese making detergents biosensor medicines Industrial Uses of Enzymes • • • • • Lactase used to convert lactose into galactose for lactose intolerant Amylase, glucose isomerase and glucoamylase are used to convert starch into high fructose syrup Pectinase replaces harsh toxic chemicals in preparing cotton for dyeing Cellulase used to “stonewash” denim and to break down wood chips into paper pulp Medical uses: proteases called plaminogen activators used to break down blood clots Control of enzyme function: I. Inhibition prevent the substrate from binding the active site a. competitive inhibitor inhibitor molecule directly binds the active site and blocks substrate b. noncompetitive inhibitor does not bind the active site directly, but causes a change in conformation (shape) in the enzyme – the shape of the active site is changed Enzyme cofactors • these non-protein factors can be organic molecules (coenzymes) or inorganic ions ( Ca+2 or Zn- ) • they enhance the enzyme’s activity enzyme + coenzyme Irreversible inhibitors: poisons substrate active site Heavy metals often bond with a thiol group (S-H) present on the cysteine amino acid. Hg enzyme They can also bind ionically with COOH, and OH groups on side chains of amino acid residues. mercury binds the active site permanently and competitively inhibits the papain enzyme Mercury Poisoning • Hg is believed to bond with SAM** (S-Adenosyl methionine) making it inactive • SAM is a coenzyme used in transferring methyl groups to proteins, nucleic acids and lipids • Used in about 40 metabolic pathways **actual mechanism is unknown, but Hg causes damage to central nervous system, endocrine system and kidneys and other organs Mercury Poisoning Elemental Mercury – Vapor is bad, liquid is not readily absorbed – Vapor present in fluorescent lights, Mercury Salts – – Dissolve in water, absorbed by gastrointestinal tract • Can't break blood-brain barrier • Cause kidney damage but not neurological damage. Used in industry, coal burning mostly • Coal fired plants are largest source (mercury video) • Also comes from incineration of waste • (can be used in manufacture of high fructose corn syrup, but new technologies in the US are Hg free...hopefully our HFCS is manufactured in the US) Organic mercury – Highly toxic: dimethylmercury, few μL = death – Hg salts are put into water by industry where it reacts with organic substances and accumulates in biological species: fish, particularly tuna, shark, swordfish Lead Poisoning • Lead replaces the zinc in an metalloenzyme called ALAD. • ALAD is responsible for making hemoglobin. • Without enough hemoglobin, brain does not get enough oxygen. • Direct links between exposure to lead and reduced cognitive abilities have been established • Studies show a relationship between lead exposure and violent crime Control of enzyme function: allosteric regulation • the enzyme usually flips back and forth between active and inactive forms by itself • if activators are present, the enzyme is stabilized in the ACTIVE form • if inhibitors are present, the enzyme is stabilized in the INACTIVE form Control of enzyme function: feedback inhibition • if there is enough product present, the product can stop (inhibit) the pathway by acting as an inhibitor • the product binds to the enzyme at an allosteric site and INACTIVATES the enzyme • this way, the pathway will only make more product if there is a need to make more cooperativity • sometimes, if there is an enzyme that can bind multiple substrates, the binding of one substrate molecule can cause the whole enzyme to be ACTIVATED and more receptive to the other substrates • ex: hemoglobin can bind 4 oxygen molecules if one oxygen binds first, this makes the enzyme able to bind the other oxygens more easily Kinetics of Enzyme Activity • Substrate concentration affects activity – usually expressed using a Michaelis-Menton plot, – enzymes which generate such a plot are said to obey Michaelis-Menton kinetics What is Vmax? • • • • Vmax is maximum rate for an enzyme catalyzed reaction can It is a limit of the enzyme. At the point when all of the active sites are engaged with substrate, Vmax has been reached. Only increasing the amount of enzyme will increase Vmax Kinetics of Enzyme Activity • E + S ES P + E • Michaelis and Menton studied enzyme kinetics – Results: an easier way to determine enzyme catalyzed reaction rate: – Km = Michaelis-Menton constant • • • • E = enzyme S = substrate ES = enzyme-subrate complex P = Product V [S ] m a x r a te v • mixture of rate constants for K [S ] forward and reverse rxns m – Vmax = theoretical max rate limited by amount of enzyme. Michaelis-Menton Plot • 3 regions – Low [S]: First order wrt to [S] – High [S]: Zero order, b/c rxn rate is limited by amount of enzyme – Middle [S]: mixed order What is Km? • Mixture of rate constants k –E 1 k2 ES k3 k2 k3 k P+E • Km is independent of the substrate concentration • Experimentally determined • Value varies with kind of substrate, temp and pH • Higher Km = lower enzyme activity • At low [S]: – [S] << Km – Rate is proportional to substrate conc. As expected for first order • At high [S] – [S] >> Km – Rate = Vmax V [S ] m a x r a te v K [S ] m Determining Km and Vmax • If … V [S ] m a x r a te v K [S ] m • When v = ½ Vmax • Km = [S] – So you can read this from the Michaelis-Menton Plot • Which enzyme has a lower Km? – Hexokinase • Which enzyme has a higher affinity for glucose? – Hexokinase • So: lower Km = faster rate There is only a small amount of hexokinase in the blood. It is very sensitive to glucose. When glucose concentrations are high, glucokinase, of which there is much more, can pick up the slack. • Why have 2 enzymes for converting sugar? • At high [glucose]: – glucokinase can speed up rapidly storing glucose in liver Another way: Lineweaver-Burke Plot • Take the inverse of both sides: V [S ] m a x r a te v K [S ] m 1 K 1 m 1 v V [] S V m a x m a x y= m • Graph of 1/rate vs. 1/[S] gives a line – Slope = Km/Vmax – Y-intercept = 1/Vmax x+ b Lineweaver-Burke Plot • Graph of 1/rate vs. 1/[S] gives a line – Slope = Km/Vmax – Y-intercept = 1/Vmax Competitive Inhibitor • Competitive Inhibition: – Takes more [S] to overcome competitive inhibitor, but eventually it is overcome. – Km changes b/c it appears that the enzyme has less affinity for the substrate, Vmax stays the same Non-competitive Inhibitor • Reduced number of active sites – Vmax changes • Km stays the same – because for the enzyme that works, there is the same affinity for the substrate as their was when the non-competitive inhibitor was present. There is just less enzyme. • Non-competitive inhibition cannot be overcome by adding more substrate • http://wiz2.pharm.wayne.edu/biochem/enz. html