ENZYMES. A protein with catalytic properties due to its power of specific activation Paul Billiet ODWS

Transcription

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

2 HISTORY of Enzymes As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified.

3 In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur came to the conclusion that this fermentation was catalyzed by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells.

4 In 1878 German physiologist Wilhelm Kühne ( ) first used the term enzyme, which comes from Greek ενζυµον "in leaven", to describe this process. The word enzyme was used later to refer to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity produced by living organisms. In 1897 Eduard Buchner began to study the ability of yeast extracts that lacked any living yeast cells to ferment sugar. In a series of experiments at the University of Berlin, he found that the sugar was fermented even when there were no living yeast cells in the mixture. He named the enzyme that brought about the fermentation of sucrose "zymase". In 1907 he received the Nobel Prize in Chemistry for his biochemical research and his discovery of cell-free fermentation". Following Buchner's example;

5 Enzymes Enzymes are Biomolecules that catalyze, increase the rates of chemical Reactions. Almost all enzymes are proteins. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products.

6 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.

7 Reaction pathway

8 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

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

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

11 The active site H.PELLETIER, M.R.SAWAYA ProNuC Database 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

12 Cofactors An additional nonprotein 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)

13 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

14 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

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

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

17 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)

18 The Induced Fit Hypothesis Hexokinase (a) without (b) with glucose substrate This explains the enzymes that can react with a range of substrates of similar types

19 Factors affecting Enzymes substrate concentration ph temperature inhibitors

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

21 Substrate concentration: Enzymic reactions V max Reaction velocity Substrate concentration Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. If you alter the concentration of the enzyme then V max will change too.

22 The effect of ph Optimum ph values Enzyme activity Trypsin Pepsin ph

23 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.

24 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.

25 The effect of temperature Enzyme activity Q10 Denaturation Temperature / C

26 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

27 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.

28 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.

29 The effect of enzyme inhibition Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories.

30 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. E + I Reversible reaction EI Enzyme inhibitor complex

31 The effect of enzyme inhibition Succinate Fumarate + 2H + + 2e - Succinate dehydrogenase CH 2 COOH COOH CHCOOH CH 2 COOH CH 2 COOH Malonate CHCOOH

32 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.

33 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