E N Z Y M E S What are they? What do they do? How do they work? What can affect how an enzyme works? Enzyme class

What are enzymes? Enzymes are __________ (tertiary and quaternary structures) proteins

DEFINITION An enzyme is a biological catalyst. This means it is a protein (biological molecule) that speeds up chemical reactions (a catalyst). A substrate is the molecule on which an enzyme acts.

Enzymes… Have names that usually end in -_____. -Sucrase -Lactase -Maltase - ase

ENZYME CLASS/NOMENCLATURE Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases Example: [EC 1.2.3.4] an oxidoreductase

ENZYME CLASS/NOMENCLATURE All enzymes will have their unique EC number. The first three numbers are the class, sub-class and sub-subclass. The last is the serial number for the enzyme in its subclass. BCH3101

ENZYME CLASS

BCH3101

BCH3101

WHAT ENZYMES DO Enzymes act in chemical reactions that build up (join simple molecules to make a bigger, complex compound) or break down (digest) big complex molecules to small simple substances. Examples: Amino acids are joined to make proteins (building up or anabolic reactions) Starch is broken down to glucose (breaking down or catabolic reactions)

SOME TERMS TO REMEMBER SUBSTRATE: the molecule upon which an enzyme acts. Example: The enzyme amylase breaks down starch. So starch is the substrate for amylase. ACTIVE SITE (of the enzyme) part of the enzyme that binds to the substrate during a chemical reaction. PRODUCT: the molecule obtained as a result of the enzyme’s action. RATE OF REACTION: how an enzymes works. It is measured by recording either the disappearance of substrate or the appearance of product. In other words, it records the changes in concentration of either the substrate or the product as the reaction proceeds.

PROPERTIES OF ENZYMES Enzymes are specific. They will react only with a one given type of substrate. Example: an enzyme that breaks down proteins will not break down fats Enzymes are affected by extreme temperatures and pH. Each enzyme has an optimum pH and temperature at which it works best.

PROPERTIES OF ENZYMES Enzymes are not affected by the chemical reaction they help take place. - same enzyme can be reused again - a small number of enzymes can carry out a great number of chemical reactions to produce a lot of products Enzymes are affected by poisons, such as toxins and heavy metals, and by high concentrations of salts

HOW ENZYMES WORK A) lock and key model: the substrate has a similar shape to the one in the active site – an enzyme-substrate complex forms – a product is obtained and the unchanged enzyme is free to start again.

Enzymes… are ________ for what they will catalyze. fit with substrate like a ____ and ____. specific lock key

B) induced fit model The substrate does not have a similar shape to the active site in the enzyme; instead, when the enzyme approaches the substrate, the enzyme’s shape changes so that the substrate can now “fit” in the active site.

Characteristics of Enzymes? speed up chemical reactions are required in minute amounts are highly specific in their action are affected by temperature are affected by pH Some catalyse reversible reactions Some require co-enzymes Are inhibited by inhibitors

Enzymes… …are _______. They are not consumed (used up) in the reactions they catalyze. reusable

(1) Enzymes Speed up chemical reactions Activation Energy with enzyme Activation Energy without enzyme Substrate Products: Energy Time By lowering the activation energy needed to start the reaction.

Enzymes catalyze reactions by weakening chemical bonds, which ________ activation energy. lowering

(2) Enzymes are required in minute amounts Chemically unchanged Sucrase Sucrose Glucose + Fructose They remain chemically unchanged after catalysing the reactions. The same enzyme molecules can be reused over again. Therefore, only a small amount of enzyme is required to catalyse a large number of reactions

(3) Enzymes are highly specific Starch Maltose Amylase Maltose Glucose + Glucose Maltase Each chemical reaction is catalysed by its own specific, unique enzyme. This is due to every enzyme’s specific 3-d configuration. How the shape of an enzyme affects its function can be explained by the “LOCK & KEY HYPOTHESIS”.

FACTORS AFFECTING ENZYME-CONTROLLED REACTIONS Temperature pH Cofactors and coenzymes (these increase the rate of reaction if present, they are necessary for the enzyme to work; e.g. vitamins) Concentration of substrate Concentration of enzymes Inhibitors

Factors That Influence Enzyme Activity Temperature pH Cofactors & Coenzymes Inhibitors

HOW TEMPERATURE AFFECTS ENZYMES At low temperatures, enzymes are inactive or work very slowly. As the temperature increases, enzymes become more active and the rate of reaction increases until the optimum temperature, when the highest rate of reaction is achieved. At higher temperatures, the enzymes are denatured (destroyed) so they can no longer bind to the substrate and no reaction takes place. HOW TEMPERATURE AFFECTS ENZYMES

Very high or very low pH affects how enzymes work, as extreme pH will denature the enzyme. Remember each type of enzyme has its own optimum pH at which it works best. HOW pH AFFECTS ENZYMES

pH and temperature of different enzymes

Substrate concentration The rate of an enzyme-controlled reaction increases as the substrate concentration increases, until the enzyme is working at full capacity At this point, the enzyme molecules reach their turnover number and assuming that all other conditions such as temperature are ideal, the only way to increase the speed of the reaction even more is to add more enzymes

Enzyme concentration In any reaction catalyzed by an enzyme, the number of enzyme molecules present is very much smaller than the number of substrate molecules. When an abundant supply of substrate is available, the rate of reaction is limited by the number of enzyme molecules present. In this situation, increasing the enzyme concentration increases the rate of reaction.

Inhibitors

Co-factor and co-enzyme Michaelis-Menten and Lineweaver-Burke Plot

Apoenzyme An enzyme that requires a cofactor but does not have one bound An apoenzyme is an inactive enzyme, activation of the enzyme occurs upon binding of an organic or inorganic cofactor

Holoenzyme An apoenzyme together with its cofactor A holoenzyme is complete and catalytically active

cofactor non-protein chemical compound that is bound to a protein and is required for the protein's biological activity.

Cofactor Examples of some enzymes that require metal ions as cofactors is shown in the table below  cofactor  enzyme or protein  Zn++  carbonic anhydrase  alcohol dehydrogenase  Fe+++ or Fe++  cytochromes, hemoglobin  ferredoxin  Cu++ or Cu+  cytochrome oxidase  K+ and Mg++  pyruvate phosphokinase

Coenzymes coenzymes are organic molecules that are required by certain enzymes to carry out catalysis They bind to the active site of the enzyme and participate in catalysis but are not considered substrates of the reaction.

coenzymes in group transfer reactions  abbreviation  entity transfered  nicotine adenine dinucelotide  NAD - partly composed of niacin  electron (hydrogen atom)  nicotine adenine dinucelotide phosphate  NADP -Partly composed of niacin  flavine adenine dinucelotide  FAD - Partly composed of riboflavin (vit. B2)  coenzyme A  CoA  Acyl groups   coenzymeQ  CoQ  electrons (hydrogen atom)  thiamine pyrophosphate  thiamine (vit. B1)  aldehydes  pyridoxal phosphate  pyridoxine (vit B6)  amino groups  biotin  carbon dioxide  carbamide coenzymes  vit. B12  alkyl groups

Michaelis-Menten http://www.youtube.com/watch?v=q94TCTSXyv8

Enzyme Kinetics Equation

The Michaelis-Menten Equation Enzyme catalyzed reactions can be described mathematically At high concentration of substrate, the E is saturated with S and the reaction rate is independent of the substrate concentration (zero order with respect to S). The maximum velocity is vmax At low concentrations of substrate - the reaction is first order with respect to S and first order with respect to E)

BCH3101

Michaelis-Menten: Equation

Lineweaver–Burk plot The y-intercept of such a graph is equivalent to the inverse of Vmax The x-intercept of the graph represents −1/Km Quick, visual impression of the different forms of enzyme inhibition Lineweaver, H and Burk, D. (1934). "The Determination of Enzyme Dissociation Constants". Journal of the American Chemical Society 56 (3): 658–666.

Michaelis-Menten vs Lineweaver–Burk plot Where:-  V is the reaction velocity (reaction rate) Km is the Michaelis–Menten constant Vmax is the maximum reaction velocity [S] is the substrate concentration

Inhibitors

Michaelis-Menten : Inhibitors

Lineweaver–Burk : Inhibitors

Lineweaver–Burk : Inhibitors

Non-competitive Inhibitors uncompetitive Inhibitors

Enzyme Activity Specific Activity is the number of units of activity per amount of total protein. Ex. A crude cell lysate might have a specific activity of 0.2 units/mg or ml protein upon which purification may increase to 10 units/mg or ml protein. One unit would be the rate formation of one μmol product per minute at a specific pH and temperature with a substrate concentration much greater than the value of Km.