ENZYMES A protein with catalytic properties due to its power of specific activation

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

Making reactions go faster Increasing the temperature make molecules move faster 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”

An enzyme controlled pathway Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.

Enzyme structure Enzymes are proteins They have a globular shape A complex 3-D structure Human pancreatic amylase © Dr. Anjuman Begum

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

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 IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

The substrate The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site

The Lock and Key Hypothesis Fit between the substrate and the active site of the enzyme is exact Like a key fits into a lock very precisely The key is analogous to the enzyme and the substrate analogous to the lock. Temporary structure called the enzyme-substrate complex formed Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate

The Lock and Key Hypothesis Enzyme may be used again Enzyme-substrate complex E S P Reaction coordinate

The Lock and Key Hypothesis This explains enzyme specificity This explains the loss of activity when enzymes denature

The Induced Fit Hypothesis Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation The active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

The Induced Fit Hypothesis Hexokinase (a) without (b) with glucose substrate http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html This explains the enzymes that can react with a range of substrates of similar types

Factors affecting Enzymes substrate concentration pH temperature inhibitors

Substrate concentration: Non-enzymic reactions Reaction velocity Substrate concentration The increase in velocity is proportional to the substrate concentration

Substrate concentration: Enzymic reactions Reaction velocity Substrate concentration Vmax 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.

The effect of pH Pepsin: the chief digestive enzyme in the stomach, which breaks down proteins into polypeptides Optimum pH values Enzyme activity Trypsin Pepsin pH 1 3 5 7 9 11 Trypsin a digestive enzyme that breaks down proteins in the small intestine. It is secreted by the pancreas in an inactive form, trypsinogen

The effect of pH Extreme pH levels will produce denaturation The structure of the enzyme is changed The active site is distorted and the substrate molecules will no longer fit in it 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 This change in ionisation will affect the binding of the substrate with the active site.

The effect of temperature Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature. For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature) Enzyme-controlled reactions follow this rule as they are chemical reactions BUT at high temperatures proteins denature The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation.

The effect of temperature Temperature / °C Enzyme activity 10 20 30 40 50 Q10 Denaturation

The effect of temperature For most enzymes the optimum temperature is about 30°C Many are a lot lower, cold water fish will die at 30°C because their enzymes denature A few bacteria have enzymes that can withstand very high temperatures up to 100°C Most enzymes however are fully denatured at 70°C

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.

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.

The effect of enzyme inhibition 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. Enzyme inhibitor complex Reversible reaction E + I EI

The effect of enzyme inhibition 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. These can be removed by using a chelating agent such as EDTA.

Applications of inhibitors Negative feedback: end point or end product inhibition Poisons snake bite, plant alkaloids and nerve gases. Medicine antibiotics

Enzyme Classification International Classification of Enzymes by the International Classification Commission in 1864. Enzymes are substrate specific and are classified according to the reaction they catalyze. Enzyme Nomenclature, 1992, Academic Press, San Diego, California, ISBN 0-12-227164-5. http://www.chem.qmul.ac.uk/iubmb/enzyme/

Enzyme Classification Enzymes can be classified into six main classes: - Oxidoreductases: catalyze the oxidation and reduction e.g. CH3CH2OH → CH3CHO+H+ - Transferases: catalyze the transfer of a functional group (e.g. a methyl or phosphate group) from one molecule (called the donor) to another (called the acceptor). A–X + B → A + B–X - Hydrolases:catalyze the hydrolysis of a chemical bond. A–B + H2O → A–OH + B–H, e.g. peptide bond

Enzyme Classification - Lyases: catalyze the breaking of various chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond or a new ring structure e.g. CH3COCO-OH → CH3COCHO (dehydratase) - Isomerases: catalyze the interconversion of isomers. e.g.phosphoglucose isomerase that converts glucose-6-phosphate to fructose-6-phosphate. - Ligases: catalyze the joining of two molecules by forming a new chemical bond.