1 Enzymes

2 What Are Enzymes? Proteins (Most enzymes are Proteins (tertiary and quaternary structures) CatalystAct as Catalyst to accelerates a reaction Not permanentlyNot permanently changed in the process

3Enzymes catalyzeAre specific for what they will catalyze ReusableAre Reusable aseEnd in –ase-Sucrase-Lactase-Maltase

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

6 How do enzymes Work? weakening bonds which lowers activation energy Enzymes work by weakening bonds which lowers activation energy

7Enzymes Free Energy Progress of the reaction Reactants Products Free energy of activation Without Enzyme With Enzyme

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.

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11 Enzyme-Substrate Complex substance enzyme substrate The substance (reactant) an enzyme acts on is the substrate Enzyme Substrate Joins

12 Active Site restricted region enzyme bindssubstrateA restricted region of an enzyme molecule which binds to the substrate. Enzyme Substrate Active Site

13 Induced Fit shapeA change in the shape of an enzyme’s active site InducedInduced by the substrate

14 Induced Fit changeconfiguration enzyme’s active siteA change in the configuration of an enzyme’s active site (H+ and ionic bonds are involved). Inducedsubstrate.Induced by the substrate. Enzyme Active Site substrate induced fit

15 What Affects Enzyme Activity? Three factors:Three factors: 1.Environmental Conditions 2.Cofactors and Coenzymes 3.Enzyme Inhibitors

16 1. Environmental Conditions 1. Extreme Temperature are the most dangerous 1. Extreme Temperature are the most dangerous - high temps denature (unfold) enzyme. - high temps may denature (unfold) the enzyme. 2.pH (most like pH near neutral) 3.Ionic concentration (salt ions)

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.REES2007 Steve Cook STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

18 2. Cofactors and Coenzymes Inorganic substances (zinc, iron) vitamins enzymatic activityInorganic substances (zinc, iron) and vitamins (respectively) are sometimes need for proper enzymatic activity. Example:Example: Ironquaternary structure-hemoglobin pick up oxygen. Iron must be present in the quaternary structure - hemoglobin in order for it to pick up oxygen.

19 Two examples of Enzyme Inhibitors a. Competitive inhibitors: resemble enzyme’s normal substrate competeactive site a. Competitive inhibitors: are chemicals that resemble an enzyme’s normal substrate and compete with it for the active site. Enzyme Competitive inhibitor Substrate

20Inhibitors b.Noncompetitive inhibitors: do not enter the active sitebind to another part enzymeenzyme change its shape alters the active site Inhibitors that do not enter the active site, but bind to another part of the enzyme causing the enzyme to change its shape, which in turn alters the active site. Enzyme active site altered Noncompetitive Inhibitor Substrate

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 ODWSODWS

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. © 2007 Paul Billiet ODWSODWS

The effect of enzyme inhibition Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories. © 2007 Paul Billiet ODWSODWS

The effect of enzyme inhibition 1.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 Rever sible reactio n E + I EI © 2007 Paul Billiet ODWSODWS

The effect of enzyme inhibition Succinate Fumarate + 2H + + 2e - Succinate dehydrogenase CH 2 C OOH CHCO OH COOH CH 2 Malonate © 2007 Paul Billiet ODWSODWS

The effect of enzyme inhibition 2.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. © 2007 Paul Billiet ODWSODWS

Applications of inhibitors Negative feedback: end point or end product inhibition Poisons snake bite, plant alkaloids and nerve gases. Medicine antibiotics, sulphonamides, sedatives and stimulants © 2007 Paul Billiet ODWSODWS

Index Definition Digestive enzymes Blood clotting Complement system

Serine Protease Cutting of certain peptide bonds in other proteins Activity depends on a set of amino acid residues in the active site of the enzyme Based on nucleophilic attack of the targeted peptidic bond by a serine

Serine Amino acid alcohol as residue

Digestive enzymes Chymotrypsin, Trypsin, Elastase closely-similar structures different substrate specificities Pancreatic proteases  proenzymes trypsinogen and chymotrypsinogen  synthesized in the pancreas and secreted into the lumen of the small intestine

Mechanism

Active site

Trypsinogen-Trypsin loss of six amino acids from one end overall structures remain similar Ca++ for thermal stability activated enzyme does have more of its structure organized into sheets

Trypsin Trypsin cleaves peptide bonds on the C-terminal side of arginines and lysines  Alcaline amino acids Activation by enteropeptidase or through protoelyse Optimum pH 7 to 8

Chymotrypsinogen-Chymotrypsin Chain is clipped in four places  release of 2 dipeptides  creation of two breaks in the backbone 1-13 segment retained as part of the active enzyme, linked on by a disulfide bond Activation by trypsin

Chymotrypsin Active site: His57-Ser195-Asp102 Chymotrypsin cuts on the C- terminal side of tyrosine, phenylalanine, and tryptophan residues hydrolysis of bonds of leucyl, methionyl, asparaginyl and glutamyl residues.

Elastase cuts peptide bonds next to small, uncharged side chains such as those of alanine, serine, valine and threonin cuts collagen

Blood Clotting System Cascade of several mechanism  Involved Serine Proteases  Thrombin  Plasmin  Factor 10 a  Factor 11 b

Human Thrombin with the Amino acids of Hirudin Thrombin Inactivated Prothrombin cleaves into activated Thrombin Thrombins cuts Paraglobuline (= fibrinoplastic substance) and forms the soluble state of it Precipitation by plasmasalt in unsoluble state

Serpins Serine Protease Inhibitors inhibit the action of their respective serine protease  serine protease binds the serpin instead of its normal substrate  protease makes a cut in the serpin leading to –the formation of a covalent bond linking the two molecules –a massive allosteric change in the tertiary structure of the serpin which moves the attached protease to a site where it can be destroyed

Occurrence/ Function stop proteolytic activity blood plasma clotting complement systems where a tiny initial activating event leads to a rapidly amplifying cascade of activity

Serpin Deficiencies inherited diseases mutation in the encoding gene for serpin examples

Alpha-1-Antitrypsin Serpin for Elastase secreted by neutrophils (lung infection)  emphysema  liver damage tests are running

The Others Other Serine Proteases –Subtilisin –Acetylcholinest erase Serpinlike Molecules –Angiotensinoge n –Chicken Ovalbumin

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