Metabolism & Enzymes

From food webs to the life of a cell energy energy energy

Flow of energy through life Life is built on chemical reactions transforming energy from one form to another organic molecules  ATP & organic molecules sun organic molecules  ATP & organic molecules solar energy  ATP & organic molecules

The First Law of Thermodynamics Energy cannot be created or destroyed, only transformed. Living systems need to continually acquire and transform energy in order to remain alive.  “Free energy”: The energy available in a system to do work.

The Second Law of Thermodynamics Every time energy is transformed, the entropy (“disorder”) of the universe increases. In order to increase/maintain their internal order, living systems must process more ordered forms of matter in to less ordered ones

Living Systems are “Open” Systems Matter and energy move in to living systems from the environment. Living systems transform matter and energy and return it to the environment

Multi-Step Metabolism To increase control, living systems produce free energy in multiple-step pathways, mediated by enzyme catalysts

That’s why they’re called anabolic steroids! Metabolism Chemical reactions of life forming bonds between molecules dehydration synthesis synthesis anabolic reactions breaking bonds between molecules hydrolysis digestion catabolic reactions That’s why they’re called anabolic steroids!

Examples dehydration synthesis (synthesis) hydrolysis (digestion) enzyme + H2O hydrolysis (digestion) enzyme + H2O

Examples dehydration synthesis (synthesis) hydrolysis (digestion) enzyme hydrolysis (digestion) enzyme

Chemical reactions & energy Some chemical reactions release energy exergonic digesting polymers hydrolysis = catabolism Some chemical reactions require input of energy endergonic building polymers dehydration synthesis = anabolism digesting molecules= LESS organization= lower energy state building molecules= MORE organization= higher energy state

Endergonic vs. exergonic reactions - energy released - digestion energy invested synthesis +G -G G = change in free energy = ability to do work

Energy & life Organisms require energy to live where does that energy come from? coupling exergonic reactions (releasing energy) with endergonic reactions (needing energy) energy + + digestion synthesis energy + +

Why don’t stable polymers spontaneously digest into their monomers? What drives reactions? If reactions are “downhill”, why don’t they just happen spontaneously? because covalent bonds are stable bonds Why don’t stable polymers spontaneously digest into their monomers? starch

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

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

Reducing Activation energy Catalysts reducing the amount of energy to start a reaction Pheeew… that takes a lot less energy! uncatalyzed reaction catalyzed reaction NEW activation energy reactant product

Catalysts So what’s a cell got to do to reduce activation energy? get help! … chemical help… ENZYMES Call in the ENZYMES! G

Enzymes Biological catalysts 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

Enzymes vocabulary substrate product active site active site products 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 enzyme

Properties of enzymes Reaction specific Not consumed in reaction each enzyme works with a specific substrate chemical fit between active site & substrate H bonds & ionic bonds Not consumed in reaction single enzyme molecule can catalyze thousands or more reactions per second enzymes unaffected by the reaction Affected by cellular conditions any condition that affects protein structure temperature, pH, salinity

Naming conventions Enzymes named for reaction they catalyze 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)

In biology… Size doesn’t matter… Shape matters! 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!

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

How does it work? Variety of mechanisms to lower activation energy & speed up reaction synthesis active site orients substrates in correct position for reaction enzyme brings substrate closer together digestion active site binds substrate & puts stress on bonds that must be broken, making it easier to separate molecules

Math Skills: Gibbs Free Energy 3.1: All living systems require constant input of free energy Be able to use and interpret the Gibbs Free Energy Equation to determine if a particular process will occur spontaneously or non-spontaneously. ΔG= change in free energy (- = exergonic, + = endergonic)  ΔH= change in enthalpy for the reaction (- = exothermic, + = endothermic) T = kelvin temperature ΔS = change in entropy (+ = entropy increase, - = entropy decrease)

Spontaneity Spontaneous reactions continue once they are initiated. Non-spontaneous reactions require continual input of energy to continue.

Using the Equation To use the equation, you’ll need to be given values. Exothermic reactions that increase entropy are always spontaneous/exergonic   Endothermic reactions that decrease entropy are always non-spontaneous/endergonic. Other reactions will be spontaneous or not depending on the temperature at which they occur.

Sample Problem Determine which of the following reactions will occur spontaneously at a temperature of 298K, justify your answer mathematically:   Reaction 1: A + B → AB Δ H: +245 KJ/mol Δ S: -.02 KJ / K Reaction 2: BC→ B + C Δ H: -334 KJ/mol Δ S: +.12 KJ/K

Got any Questions?! 2007-2008

Factors that Affect Enzymes 2007-2008

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

Enzyme concentration reaction rate enzyme concentration What’s happening here?! reaction rate enzyme concentration

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

Substrate concentration What’s happening here?! reaction rate substrate concentration

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

Temperature What’s happening here?! 37° reaction rate temperature

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 = lose 3D shape (3° structure) Cold: decrease T° molecules move slower decrease collisions between enzyme & substrate

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)

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?

Metabolic Strategies Ectothermy: Conform internal temperature to environmental temperature. Endothermy: Regulate internal temperature within a narrow range. Both strategies have advantages and tradeoffs.

Body Size Considerations Smaller animals need to produce more energy per unit of mass due to increased radiation of heat into the environment.

Free Energy Considerations & Reproduction Reproductive strategies are optimized for free energy considerations. Ex. Seasonal Reproduction.

Insufficient Free Energy Production: Individuals Insufficient free energy production by individuals will lead to disease and death.

Insufficient Free Energy Production: Populations If the individuals in the population are unable to survive, the growth rate of the population will decline

Insufficient Free Energy Production: Ecosystems If the populations in an ecosystem decline, the ecosystems will decrease in complexity

Math Skills: Coefficient Q10 3.1: All living systems require constant input Be able to use and interpret the Coefficient Q10 equation: t2 = higher temperature t1 = lower temperature k2= metabolic rate at higher temperature k1= metabolic rate at lower temperature Q10 = the factor by which the reaction rate increases when the temperature is raised by ten degrees of free energy

Q10 tells us how a particular process will be affected by a 10 degree change in temperature. Most biological processes have a Q10 value between 2 and 3

Sample Problem Data taken to determine the effect of temperature on the rate of respiration in a goldfish is given in the table below. Calculate the Q10 value for this data. (°C Temperature (°C) Heartbeats per minute 20 18 25 42

pH pepsin trypsin reaction rate pH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 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

Factors affecting enzyme function pH changes in pH adds or remove H+ disrupts bonds, disrupts 3D shape disrupts attractions between charged amino acids affect 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

Salinity What’s happening here?! reaction rate salt concentration

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!

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

Compounds which regulate enzymes Inhibitors molecules that reduce enzyme activity competitive inhibition noncompetitive inhibition irreversible inhibition feedback inhibition

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.

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.

Irreversible inhibition Inhibitor permanently binds to enzyme competitor permanently binds to active site allosteric 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.

Allosteric regulation Conformational changes by regulatory molecules inhibitors keeps enzyme in inactive form activators keeps enzyme in active form Conformational changes Allosteric regulation

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

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

Whoa! All that going on in those little mitochondria! 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!

allosteric inhibitor of enzyme 1 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

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

Don’t be inhibited! Ask Questions! 2007-2008