MITOCHONDRIAMITOCHONDRIAL LABL LAB

We are alive because we make a lot of ATP We are alive because we make a lot of ATP and ATP makes (nonspontaneous) and ATP makes (nonspontaneous)

chemical reactions take placechemical reactions take place

We make about 95% of our ATP in the We make about 95% of our ATP in the mitochondriamitochondria

We will isolate mitochondria, and study one We will isolate mitochondria, and study one enzymatic step in the pathway to making enzymatic step in the pathway to making

ATPATP

Mitochondria and Mitochondria and diseasedisease::1.1. CU MED CENTER: MITOCHONDRIAL CU MED CENTER: MITOCHONDRIAL

MALFUNCTION LEADS TO PARKINSON’S DISEASE MALFUNCTION LEADS TO PARKINSON’S DISEASE (SHAKING AS YOU AGE)(SHAKING AS YOU AGE)

2.2. IF YOU RESTRICT YOUR DIET, YOU MAY LIVER IF YOU RESTRICT YOUR DIET, YOU MAY LIVER LONGER & HEALTHIER; MORE CALORIES LONGER & HEALTHIER; MORE CALORIES TAKEN IN, MORE NAD+ IS USED TO CARRY TAKEN IN, MORE NAD+ IS USED TO CARRY ELECTRONS (NOTE THAT FAD ALSO CARRIES ELECTRONS (NOTE THAT FAD ALSO CARRIES ELECTRONS). HOWEVER, NAD+ IS ALSO NEEDED ELECTRONS). HOWEVER, NAD+ IS ALSO NEEDED FOR THE SIR2 PROTEIN THAT PREVENTS AGING FOR THE SIR2 PROTEIN THAT PREVENTS AGING SYMPTOMS. THUS, LOTS OF SUGAR INTAKE, NAD+ SYMPTOMS. THUS, LOTS OF SUGAR INTAKE, NAD+ IS NOT AVAILABLE TO SIR2– YOU AGE FASTER. IS NOT AVAILABLE TO SIR2– YOU AGE FASTER.

3.3. Bad mitochondria may be related to DiabetesBad mitochondria may be related to Diabetes

4. 4. Aging Aging (less (less ATP ATP

made made by by

mitochomitochon-dria)n-dria)

How we make most of our ATP: How we make most of our ATP: AEROBIC RESPIRATIONAEROBIC RESPIRATION: :

GLUCOSE IS BROKEN DOWN (HIGH GLUCOSE IS BROKEN DOWN (HIGH ENERGY CHEMICAL BONDS ENERGY CHEMICAL BONDS

BROKEN, ATOMS REMOVED) TO BROKEN, ATOMS REMOVED) TO STRIP OFF ENERGETIC ELECTRONS.STRIP OFF ENERGETIC ELECTRONS.ENERGY FROM ELECTRONS IS USED ENERGY FROM ELECTRONS IS USED TO MAKE ATP in the MITOCHONDRIATO MAKE ATP in the MITOCHONDRIAOXYGEN (0OXYGEN (022) ACCEPTS THE SPENT ) ACCEPTS THE SPENT

(LOW ENERGY) ELECTRONS= (LOW ENERGY) ELECTRONS= AEROBICAEROBIC

PARTS OF AEROBIC PARTS OF AEROBIC RESPIRATIONRESPIRATION::1)1) GLYCOLYSISGLYCOLYSIS occurs in the cytoplasm (glucose occurs in the cytoplasm (glucose

broken in HALF to produce 2 pyruvate molecules) broken in HALF to produce 2 pyruvate molecules) (ch. 9) (some list other (ch. 9) (some list other “step” as ‘intermediate step”; moving pyruvic “step” as ‘intermediate step”; moving pyruvic acid into the mitochondrion)acid into the mitochondrion)

2)2) TCA CYCLE (or Kreb’s cycleTCA CYCLE (or Kreb’s cycle)- where what is left )- where what is left of glucose is broken all the way down to C0of glucose is broken all the way down to C02 2 and and

all the electrons are stripped offall the electrons are stripped off

3)3) Electrons are carried (by NADH or FADH2) to Electrons are carried (by NADH or FADH2) to the the electron transport chain and ATP synthaseelectron transport chain and ATP synthase where ATP is made from electron energy (ch. 10)where ATP is made from electron energy (ch. 10)

2

Part 3

Part 1.

PARTS OF AEROBIC RESPIRATION

CH. 9: CH. 9: GLYCOLYSISGLYCOLYSIS GLYCOLYSIS IS A SERIES OF 12 GLYCOLYSIS IS A SERIES OF 12

CHEMICAL REACTIONS OCCURING IN CHEMICAL REACTIONS OCCURING IN THE CYTOPLASMTHE CYTOPLASM

TAKE GLUCOSE (LIKE JET FUEL) AND TAKE GLUCOSE (LIKE JET FUEL) AND STRIPS OFF ITS ELECTRONS IN THE HIGH STRIPS OFF ITS ELECTRONS IN THE HIGH ENERGY COVALENT BONDSENERGY COVALENT BONDS

THIS BREAKS THE COVALENT BONDS THIS BREAKS THE COVALENT BONDS AND BREAKS GLUCOSE IN HALF AND BREAKS GLUCOSE IN HALF PRODUCING TWO 3 CARBON MOLECULES PRODUCING TWO 3 CARBON MOLECULES CALLED CALLED PYRUVATEPYRUVATE

LATER, ENERGY FROM THESE LATER, ENERGY FROM THESE ELECTRONS WILL BE USED TO MAKE ATPELECTRONS WILL BE USED TO MAKE ATP

ENERGETIC ELECTRONS ENERGETIC ELECTRONS TAKEN FROM GLUCOSETAKEN FROM GLUCOSE

GIVEN TO NAD+GIVEN TO NAD+WHICH CARRIES WHICH CARRIES ELECTRONS INTO ELECTRONS INTO MITOCHONDRIAMITOCHONDRIA

ELECTRONS GIVEN TO ELECTRONS GIVEN TO NAD is a REDUCTIONNAD is a REDUCTION

USE MNEMONIC: OILRIGUSE MNEMONIC: OILRIG

Ch. 10: Last 2 Parts of Aerobic Ch. 10: Last 2 Parts of Aerobic Respiration take place in the Respiration take place in the Mitochondrion. ATP made at Mitochondrion. ATP made at

inner membraneinner membrane

FOLDING MEMBRANEDOES WHAT? (HINT:

GUT FOLDS)

Last 2 Parts involve the Last 2 Parts involve the Electron Transport Chain Electron Transport Chain

(where electrons are stripped (where electrons are stripped of their energy, energy used of their energy, energy used

to pump Protons H+)to pump Protons H+)Followed by allowing H+ to Followed by allowing H+ to

move back, turning ATP move back, turning ATP Synthase to make ATP. See Synthase to make ATP. See

SUMMARY ANIMATION SUMMARY ANIMATION FROM OUR TEXTBOOK…FROM OUR TEXTBOOK…

D:\cell D:\cell biolbiol 3611\mito respiration\respir 3611\mito respiration\respiration 1418m.movation 1418m.mov

Electron transport Electron transport animation from “Virtual animation from “Virtual

cell” web site cell” web site –see –see LINK ON our Cell Lab web site LINK ON our Cell Lab web site LAST STEP: ATP SYNTHASE LAST STEP: ATP SYNTHASE

IS LIKE A LITTLE IS LIKE A LITTLE MOLECULAR TURBINE; MOLECULAR TURBINE;

TURBINE IS ROTATED BY TURBINE IS ROTATED BY MOVEMENT OF H+; THEN MOVEMENT OF H+; THEN

TURBINE MAKES ATP. TURBINE MAKES ATP. Animations:Animations:

D:\cell D:\cell biolbiol 3611\mito respiration\ 3611\mito respiration\ETCAdvanced.wmvETCAdvanced.wmv

ANIMATIONS OF ANIMATIONS OF CHEMIOSMOSISCHEMIOSMOSIS

D:\cell D:\cell biolbiol 3611\mito respiration\chemiosmosis2.swf 3611\mito respiration\chemiosmosis2.swf

D:\cell D:\cell biolbiol 3611\mito respiration\ATP SYNTHASE MBC 14_1 3611\mito respiration\ATP SYNTHASE MBC 14_1.mov.mov

D:\cell D:\cell biolbiol 3611\mito respiration\17 ELEC TRANS CHAIN.M 3611\mito respiration\17 ELEC TRANS CHAIN.MPGPG

D:\cell D:\cell biolbiol 3611\mito respiration\ 3611\mito respiration\ATPGradientAdvanced.wmvATPGradientAdvanced.wmv

((NOTE THAT SOME ANIMATIONS TALK ONLY OF THE NOTE THAT SOME ANIMATIONS TALK ONLY OF THE H+ CONCENTRATION GRADIENT, IGNORING THE H+ CONCENTRATION GRADIENT, IGNORING THE VERY IMPORTANT ELECTRICAL GRADIENT FOR THE VERY IMPORTANT ELECTRICAL GRADIENT FOR THE H+!!)H+!!)

We will study one enzyme We will study one enzyme in the TCA Cycle in the TCA Cycle

(see Ch. 10; esp figures (see Ch. 10; esp figures used here) used here)

Succinate Succinate DehydrogenaseDehydrogenase

this enzyme breaks two this enzyme breaks two chemical bonds and chemical bonds and

removes two H atoms from removes two H atoms from what is left of glucose. what is left of glucose.

Succinate Dehydrog.Is located here…

Succin DehydrogActually bindsMembrane proteins Of the Electron TransportChain

Pyruvate comes Pyruvate comes in from the in from the

cytoplasm into cytoplasm into the the

mitochondrion, mitochondrion, it is broken it is broken down in the down in the

TCA cycle to TCA cycle to C0C022 and water. and water.

We will study We will study TCA step 6…TCA step 6…

In this reaction, In this reaction, once again what once again what is left of glucose is left of glucose is broken down is broken down

further by further by breaking bonds breaking bonds

and removal of 2 and removal of 2 H atoms. FADHH atoms. FADH2 2

carries the carries the excited electrons excited electrons to the electron to the electron transport chain transport chain (to make ATP (to make ATP from electron from electron

energy)energy)

Better view of reaction… note Better view of reaction… note the two H atoms that are the two H atoms that are removed are on different removed are on different carbons and on opposite carbons and on opposite

sides (trans, not cis)sides (trans, not cis)

In the lab, the electrons are not given to FAD, but we add a dye that changes its absorbance when it takes the electrons (change in absorbance recorded by spectrophotometer)

Succinate Dehydrogenase is Succinate Dehydrogenase is an enzyme; substrate an enzyme; substrate succinate binds in the succinate binds in the

active site active site (similar to enzymes (similar to enzymes below)below)

Characteristics of Succ. Characteristics of Succ. Dehyd.Dehyd. As it breaks chemical bonds between As it breaks chemical bonds between

Carbon and Hydrogen (C-H) in succinate, Carbon and Hydrogen (C-H) in succinate, it takes the excited electrons and the it takes the excited electrons and the Hydrogen atoms (actually hydride) from Hydrogen atoms (actually hydride) from the chemical bonds and gives them to FADthe chemical bonds and gives them to FAD

FAD becomes FADHFAD becomes FADH22 FADHFADH22 transfers the electrons to the transfers the electrons to the

electron transport chain. electron transport chain. Energy from excited electrons used to Energy from excited electrons used to

make ATPmake ATP

Cont’dCont’d Succ. Dehyd. Is an Succ. Dehyd. Is an Integral Integral (?) membrane (?) membrane

protein in the inner mitochondrial membrane protein in the inner mitochondrial membrane (hard to remove from membrane, hard to study)(hard to remove from membrane, hard to study)

All other TCA cycle enzymes are All other TCA cycle enzymes are soluble soluble (located in the matrix)(located in the matrix)

If we add a “reducible dye,” the dye If we add a “reducible dye,” the dye not FADnot FAD will pick up the electronswill pick up the electrons

OILRIG: oxidation is loss of electrons, OILRIG: oxidation is loss of electrons, reduction is gain of electrons.reduction is gain of electrons.

So, dye (or FAD) is reduced, succinate is So, dye (or FAD) is reduced, succinate is oxidized to fumarateoxidized to fumarate

Cont’dCont’d Succinate dehyd.Succinate dehyd. has a size of 100,000 daltons. has a size of 100,000 daltons.

Average protein is 50,000 daltons ; how many amino Average protein is 50,000 daltons ; how many amino acids in succ dehy? (/100)acids in succ dehy? (/100)

Also contains 8 iron atoms,Also contains 8 iron atoms, Fe (iron) atoms help in the transfer of electrons from Fe (iron) atoms help in the transfer of electrons from

succinate to FAD.succinate to FAD. Has two subunits (so it has quarternary structure)Has two subunits (so it has quarternary structure) Has higher activity than any other TCA cycle enzymeHas higher activity than any other TCA cycle enzyme

Succinate Dehydrogenase is Succinate Dehydrogenase is turned on or off through turned on or off through

allosteric regulation allosteric regulation (page 144-145)….(page 144-145)…. Allosteric regulationAllosteric regulation is how the body is how the body controls an enzyme (competitive inhibition controls an enzyme (competitive inhibition is typically artificial or external to the body)is typically artificial or external to the body)

ATP or reduced coenzyme Q are allosteric ATP or reduced coenzyme Q are allosteric activators of Succ Dehydactivators of Succ Dehyd

Allosteric activators typically bind Allosteric activators typically bind somewhere between the subunits of Succ somewhere between the subunits of Succ Dehyd (not the active site) Dehyd (not the active site) to stimulateto stimulate the the enzyme activityenzyme activity

Allosteric inhibitors act similarly Allosteric inhibitors act similarly to inhibitto inhibit

Competitive InhibitionCompetitive Inhibition Inhibitor resembles Substrate Inhibitor resembles Substrate This is not how the body/cell regulates enzymes This is not how the body/cell regulates enzymes

(typically) - some medicines work this way (typically) - some medicines work this way So this method is “artificial” and used in test So this method is “artificial” and used in test

tubes to study an enzymetubes to study an enzyme The inhibitor can bind to the active site The inhibitor can bind to the active site

(preventing the normal substrate from binding) (preventing the normal substrate from binding) but the inhibitor cannot form the product but the inhibitor cannot form the product

So, both the inhibitor and Substrate So, both the inhibitor and Substrate competecompete for for the active site of the enzymethe active site of the enzyme

If the substrate is in excess, the inhibitor will not If the substrate is in excess, the inhibitor will not inhibitinhibit

Competitive Inhibitors Competitive Inhibitors resemble the normal substrate resemble the normal substrate

(but cannot be turned into (but cannot be turned into product—so they tie up product—so they tie up

enzymes by binding to their enzymes by binding to their active site)active site)

Malonate

Competitive Inhibitors resemble the normal Competitive Inhibitors resemble the normal substrate -but cannot be turned into productsubstrate -but cannot be turned into product—so they tie up enzymes by binding to their —so they tie up enzymes by binding to their active site.active site. Malonate Malonate is a molecule that looks like is a molecule that looks like succinate, but it cannot be made succinate, but it cannot be made intointo fumaric fumaric acid (product) so malonate is a competitive acid (product) so malonate is a competitive inhibitor. inhibitor. Malonate is in a Malonate is in a COMPETITIONCOMPETITION for the for the active site of the enzyme with succinate-- active site of the enzyme with succinate-- which ever is in higher concentration typically which ever is in higher concentration typically wins!wins!Some medicines are competitive inhibitorsSome medicines are competitive inhibitors

Other competitive Other competitive inhibitors…inhibitors…

Other “dibasic acids” (means that they have Other “dibasic acids” (means that they have two carboxylic acid functional groups= C00two carboxylic acid functional groups= C00 --) ) can act as competitive inhibitorscan act as competitive inhibitors

The other dibasic acids inhibit because the The other dibasic acids inhibit because the distance between the two C00distance between the two C00- - is about the is about the same as the distance in succinate.same as the distance in succinate.

The active site of succinate dehydrogenase The active site of succinate dehydrogenase must have two + charges that are separated by must have two + charges that are separated by the same distancethe same distance

Note that as long as the Note that as long as the spacing between the two – spacing between the two –

ends is ~same as in ends is ~same as in Succinate, get competitive Succinate, get competitive

inhibition. Einhibition. Even two negative ven two negative charges of pyrophosphate can charges of pyrophosphate can

act as a negative inhibitor:act as a negative inhibitor:

0 = C - C - C – C = 0

SUCCINATE DEHYDROGENASE

ACTIVE SITE- WHERE SUBSTRATE OR COMPETITIVE INHIBITORS BIND. HERE, FIND AMINO ACIDS WHERE THEIR R GROUP HAS + CHARGE

0- 0-

+ +

SUCCINATE FITS INTO ACTIVE SITE(SOME OTHER “DIBASIC ACIDS” HAVE SAME SPACING

BETWEEN NEGATIVE CHARGES)

So, we will isolate So, we will isolate mitochondria using mitochondria using

centrifugation, and study centrifugation, and study Succinate DehydrogenaseSuccinate Dehydrogenase

To Isolate Organelles, you To Isolate Organelles, you homogenize the cell and homogenize the cell and

then use centrifugtation to then use centrifugtation to Isolate the organelleIsolate the organelle

A rotor A rotor moves round moves round and round, and round, and heavy and heavy particles particles

move to the move to the bottom of the bottom of the

test tube test tube fasterfaster

Text: p. 322-Text: p. 322-323, Sixth Ed323, Sixth Ed

Differential Centrifugation

So, we will 1. isolate mitochondria from Xenopus liver and 2. follow Succinate Dehydrogenase activity by adding Succinate3. add a competitive inhibitor called Malonate to reduce Succ Dehyd activity

Enzyme KineticsEnzyme Kinetics If little substrate is around, there will be very little If little substrate is around, there will be very little

enzyme activity and the rate of the reaction will be enzyme activity and the rate of the reaction will be slowslow

If there is more substrate around, the enzyme will If there is more substrate around, the enzyme will be more active and the reaction will be fasterbe more active and the reaction will be faster

At a certain point, even if you raise the substrate At a certain point, even if you raise the substrate concentration further, the rate of the reaction will concentration further, the rate of the reaction will not increasenot increase

THIS IS SATURATION KINETICSTHIS IS SATURATION KINETICS

(page 136 to 139 in text; 6(page 136 to 139 in text; 6thth ed) ed)

Saturation at higher substrate concentrationsbecause all enzyme working as hard as they can- the enzymesAre saturated!

Rate ofReaction(slope of OD600 vs. time)

Vm = maximum velocity or Vm = maximum velocity or rate of the reactionrate of the reaction

Km = a measure of enzyme-Km = a measure of enzyme-substrate affinity (low Km substrate affinity (low Km

means high affinity)means high affinity) Obtain Vm by going over from the Y axis (rate Obtain Vm by going over from the Y axis (rate of the reaction when it first begins) to where the of the reaction when it first begins) to where the “rectangular hyperbola” levels off“rectangular hyperbola” levels off

Obtain Km by going down the Y axis to one half Obtain Km by going down the Y axis to one half Vm, then going over to the line in the graph, Vm, then going over to the line in the graph, then going down to X axis. then going down to X axis.

You can also obtain the values by using a You can also obtain the values by using a Double Reciprocal Plot (Fig. 6-12 and 6-13).Double Reciprocal Plot (Fig. 6-12 and 6-13).

= [Succinate]

Rate is initial slope for each concentration of succinate (in OD600Versus time)

DoubleReciprocalPlot