Today is Tuesday,November 4th, 2014

Pre-Class:List as many things as you can about enzymes. What do you

remember?

Think: What do we call the molecules it “works with?” Where on the enzyme does all the action happen? What can break an enzyme? Of

what are they made?

Oh yeah, get a [small] paper towel too.

In This Lesson:Metabolism and

Enzymes(Lesson 1 of 3)

Today’s Agenda

• Chemical reactions with respect to energy changes.

• Gibbs Free Energy.• Enzymes.• Enzyme functions and mechanisms.

• Where is this in my book?– Chapter 8.

By the end of this lesson…

• You should be able to distinguish between endergonic and exergonic reactions.

• You should be able to describe the features of an enzyme.

• You should be able to “use” an enzyme, as in, understand how to turn it on and turn it off.

Let’s not get ahead of ourselves…

• Challenge questions!

The Circle of…?

• The Lion King had the Circle of Life:

http://www.toymashup.com/wp-content/uploads/2012/05/circle-of-life-game.jpg

The Circle of…?

http://media-cache-ec0.pinimg.com/736x/39/46/02/3946025d932005939bca6c884b4d9001.jpg

• South Park had the Circle of Poo:

The Circle of…?• In reality, it’s all a Circle of Energy. Kind of.

The sun’s energy is converted to ATP and Organic Molecules by

plants…

…which are converted to ATP and Organic

Molecules by herbivores…

…which are converted to ATP and Organic

Molecules by carnivores…

↓ Simba? ↓

Overview

• Metabolism is really just “the chemical reactions of life.”• Anabolism

– Forming bonds between molecules.• Dehydration synthesis, synthesis of polymers.• Fun Fact: Anabolic steroids get their name from this.

• Catabolism– Breaking bonds between molecules.

• Hydrolysis, digestion, breakdown of molecules.

• Metabolism can also be considered to include regulation of enzymes as well, even if there’s no real product associated with that part of the process.

REMINDER

• Dehydration synthesis:

• Hydrolysis/digestion:

H2O

H2O

REMINDER

• Dehydration synthesis:

• Hydrolysis

Energy Release/Absorption• Reactions can further be classified by whether they

have a net release or absorption of energy.– Exergonic reactions have a net release of energy and are

associated with digestion (breaking down) of molecules:

-ΔG

Energy Release/Absorption

• Endergonic reactions have a net absorption of energy and are associated with synthesizing (building) molecules:

+ΔG

Wait…ΔG?

• ΔG is equal to the Gibbs free energy of the reaction.– Think of free energy as the ability to do work.– When there is an endergonic reaction, energy is put into

the molecules that can later be used to do work.– Exergonic reactions release energy that can be used

elsewhere.– You’ve already seen this with ATP:

• Using ATP requires the “breaking off” of a phosphate group, releasing energy.

• Rebuilding ATP from ADP requires the addition of a phosphate group, requiring energy.

Gibbs Free Energy

• Gibbs free energy is a product of thermodynamics. Most relevant to biology:– First Law: Energy is constant in the universe and reactions.– Second Law: Spontaneous reactions increase the entropy

(disorder) of the universe.• For a basic example, consider that it’s far more likely for an egg to

break into a bunch of pieces than it is for a bunch of pieces to form an egg.– See also: Humpty Dumpty.

• Obviously what cells do is an exception.

– Side Note: Don’t confuse entropy (disorder) with enthalpy (heat energy change).

Gibbs Free Energy Equation

• ΔG = ΔH – TΔS

• ΔG = Gibbs Free Energy• ΔH = Change in enthalpy (heat)• T = Temperature (in Kelvin)• ΔS = Change in entropy (disorder)

Gibbs Free Energy Equation

• Units:– ΔG and ΔH are given in heat units:• calories (cal) or kilocalories (kcal)• 1000 cal = 1 kcal

– ΔS is given as heat/Kelvin.

• Okay, practice problem time!

Gibbs Free Energy Example• If the change in free energy is -5.05 kcal for a reaction occurring

at 22 °C and the change in entropy is 100 cal/K, what is the change in enthalpy? Is the reaction endergonic or exergonic?

• 22 °C = 295 K• ΔG = ΔH – TΔS• -5.05 kcal = ΔH – (295 K)(100 cal/K)• -5.05 kcal = ΔH – 29500 cal• -5.05 kcal = ΔH – 29.5 kcal• ΔH = 24.45 kcal

– Which means this is exergonic since free energy decreases.• +ΔG = endergonic; -ΔG = exergonic• (+ΔH = endothermic; -ΔH = exothermic)

Time to Practice Independently

• Gibbs Free Energy Practice Problems worksheet

Thermodynamic ConceptsNot incredibly important for Biology but here if you’d like to know.

• When you think about it, the free energy equation is really just a relationship between enthalpy and entropy (adjusted for temperature).

• The “TΔS” part of the equation (entropy) needs to be larger than the “ΔH” part of the equation (enthalpy) to make the reaction exergonic.– An exothermic reaction (-ΔH) is not necessarily

exergonic, but it’s somewhat energetically favorable.– If ΔH is negative and ΔS is positive (increase in entropy),

the equation is VERY thermodynamically favorable.

ΔG = ΔH – TΔS

About Pi

• Did you catch the Pi in the first problem?• The hydrolysis of ATP goes by this reaction:– ATP → ADP + Pi

• Pi is the notation for a free phosphate group.– The “i” stands for “inorganic.”– “Inorganic” just means that it’s not being pulled off

some other molecule.

Coupling Reactions

• Because organisms need to, you know, live, they must couple endergonic reactions with exergonic reactions.

• To put it another way, think of what we do every day: eating.– You eat food which is then digested and broken

down to provide your body with energy.– That energy is then used to help you grow and do

things that require energy. Coupled!

Coupling Reactions

• In other words:

+ + Energy

+ +

Coupling Reactions

• One more example. Take a look at the inner membrane of the mitochondria for a great look at coupled reactions.– More on this to come later:

The Big Picture

• Keep in mind that big organic compounds with lots of chemical bonds (especially giant hydrocarbons) contain a lot of bonding energy.

• This explains why fat is such a good source of energy.• Remember triglycerides? They’re among the most

energy-containing molecules out there:

Spontaneity

• Reactions don’t just happen spontaneously, however.– Imagine immediately losing starch molecules to spontaneous

“digestion.”• Consider, for example, the thermite reaction:– Thermite is a mix of aluminum powder and iron oxide powder

(essentially rust).– It was originally used for “welding” railroad ties together out in

the wilderness.– However, it’s important to note that just mixing the substances

does not actually set off the reaction.– In the video, watch for the input of energy to “kick-start” things.

• Thermite video

Activation Energy• The use of the sparkler in the video provided the activation

energy necessary to start the reaction.– Activation energy is the amount of energy needed to destabilize a

molecule’s bonds.– Exergonic reactions can happen spontaneously, but activation

energy makes them process sloooooooooowly. Too sloooooooooowly for living things.

– ∆G is not a part of activation energy.• In graph form:

Activation Energy

• Catalysts are substances (not necessarily organic substances) that help lower activation energy.

• Enzymes are proteins that lower activation energy.

Important Distinction

• Many people realize that enzymes speed up reactions.

• It’s important to realize, however, that they do not increase the “movement” of the particles involved.

• Remember, enzymes lower activation energy.• Here’s a conceptual example:

Enzyme Function Example

• Imagine you earn an allowance of $1 per week.• You want to buy a video game system that costs $349.– How many weeks do you need?

• 349. Enjoy that.

• If the game system goes on sale for $50, how many weeks would you need?– 50.

• So you can get the system sooner, but are you earning money at a faster rate?– No.– This is how enzymes work – they lower the “threshold.”

Enzyme Details

• Enzymes are biological catalysts:– Made of protein or RNA (RNA enzyme = ribozyme).– Facilitate chemical reactions by:• Lowering activation energy to increase reaction rate.• Not being consumed in reactions.

– A single enzyme can catalyze thousands of reactions per second.

• Not changing ∆G released or required.

– Required for most biological reactions.• Reactions would take too long otherwise.

– Highly specific (thousands of different kinds in each cell)

Enzymes• Enzymes come with their own vocabulary:– The reactant which binds to an enzyme is called the substrate.

• Once bound, they are temporarily called the enzyme-substrate complex.

• Products are the substrates after the reaction.

– Exactly where on the enzyme molecule a substrate binds is called the active site.

Specificity

• What’s this about being specific?• Enzymes fit their substrates and only their substrates

through something known as the lock and key model.– Quite like a key fitting into a lock, only with hydrogen bonds.

• This is a functional but simplistic model.

Specificity

• More accurate is the induced fit model:– Just like the lock and key model, except the binding of

the substrate causes a conformational change in the enzyme that leads to an even closer fit.

– Functional groups become closer together for catalysis.

What’s the difference?

• Imagine a constrictor snake killing its prey:

• As the prey exhales, the snake coils more tightly, preventing the prey from inhaling again.

• In the same way, in the induced fit model, the substrate fits the enzyme like a key into a lock, but the binding of the substrate causes it to fit even more tightly.

http://cdn1.arkive.org/media/5C/5CA79B70-01FA-4CCB-8270-6E6AD8D9452A/Presentation.Large/Green-anaconda-constricting-ibis-prey.jpg

What’s in a name?

• Enzymes also have friendly names, usually:– Sucrase breaks down sucrose.– Proteases break down proteins.– Lipases break down lipids.– DNA Polymerase polymerizes DNA.– ATP Synthase synthesizes ATP.– Pepsin breaks down polypeptides.

• In other words, enzymes are named for their reactions.

Enzyme Mechanisms

• Without going into tremendous detail, how do enzymes actually do their jobs?

• Here are a couple of examples:– During synthesis reactions, the active site of the

enzyme orients the substrates in a correct position so as to bring them closer together.• Like a kid that puts two dolls together and says, “And now

they kiss!”

– During digestion reactions, the active site puts stress on the bonds that must be broken to facilitate breakage.

Whew. Okay, that was a lot…

• Time for, you guessed it, a POGIL!– “Yes! I LOVE POGILS!”

• Note that this one will take you through a review of enzymes and then help you explore some new concepts about them.

• We’ll be exploring those new concepts afterward, so if you don’t know a question, leave it blank for now.– Mainly these are questions concerning the factors that

affect enzyme function.

Enzyme Contest

• I need ten volunteers.• Five of you are going to play the role of an enzyme

called Splint Splittase. Guess what it does.– It splits splints (twice each, into four total pieces).

• I’ll also need five people to act as official judges.– You’ll count how many splints are split.– Remember, one splint needs to be split into four for it to

count as one.

Enzyme Contest

• There is a catch, however.• To explore the limiting factors of enzymes,

we’re going to add some details:– One of you is a control group.– One of you is going to cross your fingers.– One of you will have a lot of splints.– One of you will have only a few splints total.– One of you is going to put your hands in ice water

for a while before we start.

Factors Affecting Enzyme FunctionSummary Slide

• Enzyme Concentration• Substrate Concentration• Temperature• pH• Salinity• Activators• Inhibitors

Enzyme Concentration

• Keep in mind, enzymes aren’t intelligent beings.– No offense if your friends or family members are

enzymes.• They need to rely on chance collisions with

substrates to be effective, so:– As enzyme concentration goes up, so does

reaction rate.• More enzymes means more collisions with substrates.

Enzyme Concentration: Graph Form

• Whoa. Why the leveling-off point?– Eventually, we reach a point at which the substrate concentration limits the

enzyme’s ability to work.– Simulated by the Splint Splittase with too few splints.– If, however, we assume that the substrate is unlimited, a different pattern

emerges.

Enzyme Concentration

Reac

tion

Rate Limited substrate

Unlimited substrate

Substrate Concentration

• In contrast, if substrate concentrations get very high, the enzyme reaches a point at which reaction rate is maximized.

• So, as substrate concentration increases, reaction rate increases (until saturation).– At saturation, all enzymes’ active sites are occupied.

• This was symbolized by the Splint Splittase with a lot of splints.– I could have added a truckload more and the rate would

not have increased.

Substrate Concentration: Graph Form

Substrate Concentration

Reac

tion

Rate Saturation reached

Temperature

• All enzymes have an optimal temperature (or range of temperatures) for maximizing molecular collisions.– Remember that increased temperature is just increased molecular

kinetic energy (motion).– Human enzymes work best between 35 °C and 40°C.

• Human body temperature = 37 °C average.• This partly explains why hyperthermia (fevers) and hypothermia can be so

dangerous.

• Heat can denature enzymes by breaking ionic and hydrogen bonds, changing their shape.

• Lack of heat causes molecules to move too slowly for enzymes to function properly.– Symbolized by the Splint Splittase in ice water (since I couldn’t start

any fires).

Temperature: Graph Form

Temperature

Reac

tion

Rate

HumanEnzyme

37 °C(98.6 °F)

Thermophilic Bacteria

70 °C(158 °F)

pH

• Changes in pH can disrupt bonds and thus denature proteins just like heat.– Heat just used kinetic energy to destabilize bonds.– For pH, the addition or removal of H+ ions disrupts the

bonds by changing attraction between charged amino acids.• Most human enzymes (but not all) work between pH 6

and pH 8. Some others:– Pepsin works in the stomach (pH 2-3)– Trypsin works in the small intestine (pH 8).

• This was symbolized by the Splint Splittase with crossed fingers (since I couldn’t dump acid everywhere).

pH: Graph Form

pH

Reac

tion

Rate

Pepsin

2.5

Trypsin

8

Salinity

• Salinity changes alter the concentrations of cations (positively charged ions) and anions (negatively charged ions).– This is any salt, not just NaCl, by the way.

• This leads to denaturation too.– The Dead Sea is dead for a good reason.– Today, biologists have come to know a number of

extremophiles – organisms (usually bacteria or archaea) that live in extremely salty, hot, or acidic/basic environments.

– Also symbolized by the Splint Splittase with crossed fingers (since I can’t dump salt water everywhere).

Salinity: Graph Form

Salinity

Reac

tion

Rate

Activators• There are three main types of activators out

there:– Cofactors are small, inorganic non-proteins that

bind to and activate the enzyme.• Like how Fe is a part of hemoglobin or Mg is in

chlorophyll.• Mg, K, Ca, Zn, and Cu also are common cofactors.

– Coenzymes are small, organic non-proteins that bind temporarily or permanently near an enzyme’s active site.• Many vitamins are coenzymes:• Coenzyme A, NAD (vitamin B3: niacin), FAD (vitamin

B2: riboflavin)

– Cooperators are substrates that act as activators by changing enzyme shapes.• Often the substrate changes the shape of a multi-

subunit enzyme to kick-start the rest of it.

InhibitorsSummary Slide

• Broadly, inhibitors work against enzyme activity.• Types of inhibition:– Competitive inhibition– Noncompetitive (allosteric) inhibition

• Additional inhibition effects/details:– Irreversible inhibition– Feedback inhibition

Inhibition: Competitive• Competitive Inhibition: When there’s

another molecule that isn’t a substrate, yet can bind to the active site of an enzyme.– Penicillin, for example, blocks an enzyme used

by bacteria to make cell walls.– Antabuse (disulfiram) is a commercial drug that

treats alcoholism by inhibiting the enzyme that breaks down alcohol, leading to severe hangover and illness within 5-10 minutes after drinking.

• Competitive inhibition can be overcome by increasing substrate concentration.– In other words, substrates can out-compete

inhibitors.• Analogy: Competitive inhibition is like

someone sitting in your seat.

Inhibition: Noncompetitive• Noncompetitive (Allosteric) Inhibition:

When an inhibitor binds somewhere other than the active site and causes a conformational change that prevents binding with the normal substrate.– This is known as an allosteric inhibitor – more

to come.– Some anti-cancer drugs inhibit enzymes that

copy DNA, preventing new cell growth.– Cyanide poison permanently inhibits

Cytochrome C, an enzyme in respiration, preventing cells from making ATP.

• Analogy: Noncompetitive inhibition is like someone sitting in the row behind you putting their feet or coat on your chair.

Effects/Details of Inhibition

• Irreversible inhibition, as with cyanide, is exactly what it sounds like.– For irreversible competitive inhibition, the inhibitor

binds permanently to the active site.– For irreversible noncompetitive inhibition, the inhibitor

binds permanently to the allosteric site.• “Allo-” meaning “other” and “-steric” meaning “shape-

related.”• These inhibitors permanently change enzyme shape, as in

many nerve gases and sarin gas, and in insecticides.

Allosteric Regulation

• In addition to allosteric inhibition, there is also allosteric activation.– In other words, shape changes can activate or deactivate

an enzyme.– Activators and inhibitors stabilize these shape changes.

Feedback Inhibition

• One last dynamic associated with inhibition is feedback inhibition.– This is when the final product of a metabolic pathway

(next slide) inhibits the earlier step(s).– This inhibition prevents unnecessary accumulation of a

product.

Substance A

Substance B

Substance C

Substance D

↑Enzyme 1

Substance

E↑

Enzyme 2↑

Enzyme 3↑

Enzyme 4

Substance E Inhibits Enzyme 1

Feedback Inhibition Example

• The amino acid isoleucine is made from another amino acid, threonine.

• Isoleucine inhibits the first step in the pathway, shutting off its own production.– Isoleucine collides with the

enzyme more than the initial substrate does.

Metabolic Pathways?

• We’ll be exploring this extensively, but now’s a good time to mention that metabolic pathways are just various series of reactions associated with metabolism.

• Like we saw with feedback inhibition, pathways allow for control/regulation of reactions as well as efficiency.

• Yay evolution!

Closure

• BBC – Bitesize Enzymes video