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Chapter 8.4, 8.5 Enzymes
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Activation energy Breaking down large molecules requires an initial input of energy activation energy large biomolecules are stable must absorb energy to break bonds Need a spark to start a fire energy cellulose CO2 + H2O + heat
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Too much activation energy for life
amount of energy needed to destabilize the bonds of a molecule moves the reaction over an “energy hill” Not a match! That’s too much energy to expose living cells to! glucose 2nd Law of thermodynamics Universe tends to disorder so why don’t proteins, carbohydrates & other biomolecules breakdown? at temperatures typical of the cell, molecules don’t make it over the hump of activation energy but, a cell must be metabolically active heat would speed reactions, but… would denature proteins & kill cells
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Reducing Activation Energy
Catalysts Pheeew… that takes a lot less energy! uncatalyzed reaction catalyzed reaction NEW activation energy reactant product
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Catalysts Reduce the amount of energy to start a reaction
2 Catalysts Reduce the amount of energy to start a reaction Cells use ENZYMES to reduce activation energy Call in the ENZYMES! G
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QUESTION 1: How do enzymes speed up chemical reactions?
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Enzymes Biological catalysts 3 proteins (& RNA)
facilitate chemical reactions increase rate of reaction without being consumed reduce activation energy don’t change free energy (G) released or required required for most biological reactions highly specific thousands of different enzymes in cells control reactions of life
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Enzyme Vocabulary substrate product active site active site products
4 Enzyme Vocabulary substrate reactant which binds to enzyme enzyme-substrate complex: temporary association product end result of reaction active site enzyme’s catalytic site; substrate fits into active site active site products substrate “transition state” enzyme
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Properties of Enzymes Reaction specific Not consumed in reaction
5 Properties of Enzymes Reaction specific each enzyme works with a specific substrate chemical fit between active site & substrate forming H bonds, ionic bonds Not consumed in reaction single enzyme molecule can catalyze thousands or more reactions per second enzymes unaffected by the reaction (reusable) Affected by cellular conditions any condition that affects protein structure temperature, pH, salinity
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Naming Conventions Enzymes named for reaction they catalyze 6
sucrase breaks down sucrose proteases break down proteins lipases break down lipids DNA polymerase builds DNA adds nucleotides to DNA strand pepsin breaks down proteins (polypeptides)
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QUESTION 2: Use slides 3 – 6 to give FOUR WORDS that describe enzymes.
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In biology… Size doesn’t matter… Shape matters!
7 Lock and Key model Simplistic model of enzyme action substrate fits into 3-D structure of enzyme’ active site H bonds between substrate & enzyme like “key fits into lock” In biology… Size doesn’t matter… Shape matters!
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Induced Fit model More accurate model of enzyme action
8 Induced Fit model More accurate model of enzyme action 3-D structure of enzyme fits substrate substrate binding cause enzyme to change shape leading to a tighter fit “conformational change” bring chemical groups in position to catalyze reaction
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QUESTION 3: What is the difference between the lock and key model and the induced fit model? Which model is more accurate?
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9 How do enzymes lower Ea? Variety of mechanisms to lower activation energy & speed up reaction 1) In synthesis, active site orients substrates in correct position for reaction enzyme brings substrate closer together 2) In digestion, active site puts stress on bonds that must be broken, making it easier to separate molecules 3) Providing a favorable microenvironment (ex: pH)
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Got any Questions?!
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Factors that Affect Enzymes
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Factors Affecting Enzyme Function
Enzyme concentration Substrate concentration Temperature pH Salinity Activators Inhibitors Living with oxygen is dangerous. We rely on oxygen to power our cells, but oxygen is a reactive molecule that can cause serious problems if not carefully controlled. One of the dangers of oxygen is that it is easily converted into other reactive compounds. Inside our cells, electrons are continually shuttled from site to site by carrier molecules, such as carriers derived from riboflavin and niacin. If oxygen runs into one of these carrier molecules, the electron may be accidentally transferred to it. This converts oxygen into dangerous compounds such as superoxide radicals and hydrogen peroxide, which can attack the delicate sulfur atoms and metal ions in proteins. To make things even worse, free iron ions in the cell occasionally convert hydrogen peroxide into hydroxyl radicals. These deadly molecules attack and mutate DNA. Fortunately, cells make a variety of antioxidant enzymes to fight the dangerous side-effects of life with oxygen. Two important players are superoxide dismutase, which converts superoxide radicals into hydrogen peroxide, and catalase, which converts hydrogen peroxide into water and oxygen gas. The importance of these enzymes is demonstrated by their prevalence, ranging from about 0.1% of the protein in an E. coli cell to upwards of a quarter of the protein in susceptible cell types. These many catalase molecules patrol the cell, counteracting the steady production of hydrogen peroxide and keeping it at a safe level. Catalases are some of the most efficient enzymes found in cells. Each catalase molecule can decompose millions of hydrogen peroxide molecules every second. The cow catalase shown here and our own catalases use an iron ion to assist in this speedy reaction. The enzyme is composed of four identical subunits, each with its own active site buried deep inside. The iron ion, shown in green, is gripped at the center of a disk-shaped heme group. Catalases, since they must fight against reactive molecules, are also unusually stable enzymes. Notice how the four chains interweave, locking the entire complex into the proper shape. catalase
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Enzyme concentration QUESTION 4: IN YOUR OWN WORDS,
describe what this graph is showing in a complete sentence. What’s happening here?! reaction rate enzyme concentration
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Factors affecting enzyme function
10 Factors affecting enzyme function Enzyme concentration as enzyme = reaction rate more enzymes = more frequently collide with substrate reaction rate levels off substrate becomes limiting factor not all enzyme molecules can find substrate Why is it a good adaptation to organize the cell in organelles? Sequester enzymes with their substrates! enzyme concentration reaction rate
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Substrate concentration
QUESTION 5: IN YOUR OWN WORDS, describe what this graph is showing in a complete sentence. What’s happening here?! reaction rate substrate concentration
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Factors affecting enzyme function
11 Factors affecting enzyme function Substrate concentration as substrate = reaction rate more substrate = more frequently collide with enzyme reaction rate levels off all enzymes have active site engaged enzyme is saturated maximum rate of reaction Why is it a good adaptation to organize the cell in organelles? Sequester enzymes with their substrates! substrate concentration reaction rate
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Temperature QUESTION 6: IN YOUR OWN WORDS,
describe what this graph is showing in a complete sentence. What’s happening here?! 37° reaction rate temperature
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Factors affecting enzyme function
12 Factors affecting enzyme function Temperature Optimum T° greatest number of molecular collisions human enzymes = 35°- 40°C (body temp = 37°C) Heat: increase beyond optimum T° increased energy level of molecules disrupts bonds in enzyme & between enzyme & substrate H, ionic = weak bonds denaturation = protein loses 3D shape (3° structure) Cold: decrease below optimum T° molecules move slower decreased collisions between enzyme & substrate
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Enzymes and temperature
13 Enzymes and temperature Different enzymes function in different organisms in different environments hot spring bacteria enzyme human enzyme 37°C 70°C reaction rate temperature (158°F)
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How do ectotherms do it? Enzymes work within narrow temperature ranges. Ectotherms, like snakes, do not use their metabolism extensively to regulate body temperature. Their body temperature is significantly influenced by environmental temperature. Desert reptiles can experience body temperature fluctuations of ~40°C (that’s a ~100°F span!). What mechanism has evolved to allow their metabolic pathways to continue to function across that wide temperature span?
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pH QUESTION 7: IN YOUR OWN WORDS, describe what this graph is showing
in a complete sentence. What’s happening here?! pepsin trypsin pepsin reaction rate trypsin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pH
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Factors affecting enzyme function
14 Factors affecting enzyme function pH changes in pH adds or remove H+ disrupts bonds, disrupts 3D shape disrupts attractions between charged amino acids affects 2° & 3° structure denatures protein optimal pH? most human enzymes = pH 6-8 depends on localized conditions pepsin (stomach) = pH 2-3 trypsin (small intestines) = pH 8 7 2 1 3 4 5 6 8 9 10 11
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Salinity QUESTION 8: IN YOUR OWN WORDS,
describe what this graph is showing in a complete sentence. What’s happening here?! reaction rate salt concentration
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Factors affecting enzyme function
15 Factors affecting enzyme function Salt concentration changes in salinity adds or removes cations (+) & anions (–) disrupts bonds, disrupts 3D shape disrupts attractions between charged amino acids affect 2° & 3° structure denatures protein enzymes intolerant of extreme salinity Dead Sea is called dead for a reason!
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QUESTION 9: What does the following words mean in everyday language
QUESTION 9: What does the following words mean in everyday language? - “regulate”/”regulation” - “activate” - “inhibit” - “competitive” - “irreversible” - “feedback”
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Compounds which help enzymes
16 Compounds which help enzymes Fe in hemoglobin Activators cofactors non-protein, small inorganic compounds & ions Mg, K, Ca, Zn, Fe, Cu bound within enzyme molecule coenzymes non-protein, organic molecules bind temporarily or permanently to enzyme near active site many vitamins NAD (niacin; B3) FAD (riboflavin; B2) Coenzyme A Hemoglobin is aided by Fe Chlorophyll is aided by Mg Mg in chlorophyll
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QUESTION 10: Compare and contrast cofactors and coenzymes.
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Compounds which regulate enzymes
17 Compounds which regulate enzymes Inhibitors molecules that reduce enzyme activity competitive inhibition noncompetitive inhibition (both could be irreversible) feedback inhibition
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Competitive Inhibitor
18 Competitive Inhibitor Inhibitor & substrate “compete” for active site penicillin blocks enzyme bacteria use to build cell walls disulfiram (Antabuse) treats chronic alcoholism blocks enzyme that breaks down alcohol severe hangover & vomiting 5-10 minutes after drinking Overcome by increasing substrate concentration saturate solution with substrate so it out-competes inhibitor for active site on enzyme Ethanol is metabolized in the body by oxidation to acetaldehyde, which is in turn further oxidized to acetic acid by aldehyde oxidase enzymes. Normally, the second reaction is rapid so that acetaldehyde does not accumulate in the body. A drug, disulfiram (Antabuse) inhibits the aldehyde oxidase which causes the accumulation of acetaldehyde with subsequent unpleasant side-effects of nausea and vomiting. This drug is sometimes used to help people overcome the drinking habit. Methanol (wood alcohol) poisoning occurs because methanol is oxidized to formaldehyde and formic acid which attack the optic nerve causing blindness. Ethanol is given as an antidote for methanol poisoning because ethanol competitively inhibits the oxidation of methanol. Ethanol is oxidized in preference to methanol and consequently, the oxidation of methanol is slowed down so that the toxic by-products do not have a chance to accumulate.
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Non-Competitive Inhibitor
19 Non-Competitive Inhibitor Inhibitor binds to site other than active site allosteric inhibitor binds to allosteric site causes enzyme to change shape conformational change active site is no longer functional binding site keeps enzyme inactive some anti-cancer drugs inhibit enzymes involved in DNA synthesis stop DNA production stop division of more cancer cells cyanide poisoning irreversible inhibitor of Cytochrome C, an enzyme in cellular respiration stops production of ATP Basis of most chemotherapytreatments is enzyme inhibition. Many health disorders can be controlled, in principle, by inhibiting selected enzymes. Two examples include methotrexate and FdUMP, common anticancer drugs which inhibit enzymes involved in the synthesis of thymidine and hence DNA. Since many enzymes contain sulfhydral (-SH), alcohol, or acid groups as part of their active sites, any chemical which can react with them acts as a noncompetitive inhibitor. Heavy metals such as silver (Ag+), mercury (Hg2+), lead ( Pb2+) have strong affinities for -SH groups. Cyanide combines with the copper prosthetic groups of the enzyme cytochrome C oxidase, thus inhibiting respiration which causes an organism to run out of ATP (energy) Oxalic and citric acid inhibit blood clotting by forming complexes with calcium ions necessary for the enzyme metal ion activator.
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QUESTION 11: What is the difference between a competitive inhibitor and a non-competitive (allosteric) inhibitor?
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Irreversible Inhibition
20 Irreversible Inhibition Inhibitor (either competitive or noncompetitive) permanently binds to enzyme competitor permanently binds to active site allosteric (non-competitor) permanently binds to allosteric site permanently changes shape of enzyme nerve gas, sarin, many insecticides (malathion, parathion…) cholinesterase inhibitors - doesn’t breakdown the neurotransmitter, acetylcholine Another example of irreversible inhibition is provided by the nerve gas diisopropylfluorophosphate (DFP) designed for use in warfare. It combines with the amino acid serine (contains the –SH group) at the active site of the enzyme acetylcholinesterase. The enzyme deactivates the neurotransmitter acetylcholine. Neurotransmitters are needed to continue the passage of nerve impulses from one neurone to another across the synapse. Once the impulse has been transmitted, acetylcholinesterase functions to deactivate the acetycholine almost immediately by breaking it down. If the enzyme is inhibited, acetylcholine accumulates and nerve impulses cannot be stopped, causing prolonged muscle contration. Paralysis occurs and death may result since the respiratory muscles are affected. Some insecticides currently in use, including those known as organophosphates (e.g. parathion), have a similar effect on insects, and can also cause harm to nervous and muscular system of humans who are overexposed to them.
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Allosteric regulation
21 Allosteric regulation Conformational changes by regulatory molecules inhibitors keeps enzyme in inactive form activators keeps enzyme in active form Conformational changes Allosteric regulation
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Metabolic pathways A B C D E F G A B C D E F G
22 Metabolic pathways A B C D E F G A B C D E F G enzyme 1 enzyme 3 enzyme 2 enzyme enzyme 4 enzyme 5 enzyme 6 Chemical reactions of life are organized in pathways divide chemical reaction into many small steps artifact of evolution efficiency intermediate branching points control = regulation
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Whoa! All that going on in those little mitochondria!
23 Efficiency Organized groups of enzymes enzymes are embedded in membrane and arranged sequentially Link endergonic & exergonic reactions Whoa! All that going on in those little mitochondria!
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allosteric inhibitor of enzyme 1
24 Feedback Inhibition Regulation & coordination of production product is used by next step in pathway final product is inhibitor of earlier step allosteric inhibitor of earlier enzyme feedback inhibition no unnecessary accumulation of product A B C D E F G enzyme 1 enzyme 2 enzyme 3 enzyme 4 enzyme 5 enzyme 6 X allosteric inhibitor of enzyme 1
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Feedback inhibition Example
threonine Feedback inhibition Example synthesis of amino acid, isoleucine from amino acid, threonine isoleucine becomes the allosteric inhibitor of the first step in the pathway as product accumulates it collides with enzyme more often than substrate does isoleucine
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Don’t be inhibited! Ask Questions!
Ask now or forever hold your peace!!!!!!!!! Don’t be inhibited! Ask Questions! QUESTION 12: Write down questions, vocabulary, or concepts that you are unclear about. Be prepared to share with me (Ms. Lin) so that we can go over it as a class!
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Cooperativity Substrate acts as an activator
substrate causes conformational change in enzyme induced fit favors binding of substrate at 2nd site makes enzyme more active & effective hemoglobin Hemoglobin 4 polypeptide chains can bind 4 O2; 1st O2 binds now easier for other 3 O2 to bind
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