Fig 2.15

Cellular Energy

Chapter 3: Enzyme function and pathways in energy metabolism

AmanText Box

Metabolic Reactions

Types of reactions: Hydrolysis (A-B + H2O A-OH + H-B), opp. condensation Phosphorylation/dephosphorylation (ADP + Pi ATP + H20) Oxidation-reduction reactions (H2 2H+ + 2e-)

Metabolism is the sum of all chemical reactions in the cell

Reactant

Product

Principles of chemical equilibrium and the law of mass action dictate the direction of reversible reactions and the eventual concentrations of reactants and products

Fig 3.2 aA + bB cC + dD

[C]c [D]d

[A]a [B]b K =

Enzymes Activation energy The energy required to go through the transition state of a reaction

Fig 3.3

The height of the activation energy barrier, along with temperature and the concentrations of reactants and products, dictate the rate of chemical reactions

Enzymes are protein catalysts that increase the rate of biochemical reactions by reducing the activation energy (usually by a factor of 105 to 1017!)

Nearly all reactions in the body are catalyzed, because they would otherwise occur far too slowly

Enzymes Enzymes are not themselves altered by the reaction they catalyze The reactant bound by an enzyme is called a substrate

Fig 3.6

Enzymes are specific to their particular substrates, and can catalyze forward and reverse reactions

Fig 3.7

Rates of enzyme reactions (V) depend upon:

3) Enzyme concentration [E]

(maximum rates = Vmax = [E]*kcat)

2) Enzyme activity (i.e., inherent catalytic rate of the protein, kcat)

1) Concentrations of substrates [S] and products [P]

(based on the chemical equilibrium, and the propensity to form enzyme-substrate (ES) complexes)

Increasing ES complexes

Enzyme kinetics

Fig 3.8

= Vmax

AmanText Boxas substrate concentration increases, the reaction rate increases

AmanTypewritten Text- the greater the concentration of enzyme the greater the reaction rate

4) The affinity of the enzyme for its substrate

Enzyme kinetics

Fig 3.9 Michaelis-Mentenequa/on:

Reac/onrate(V)= [S]VmaxKm+[S]

WhereKmisthe[S]atwhichV=Vmax

5) Temperature, pH, and other physical factors

Enzymes with higher affinity have lower Km

Rates of enzyme reactions (V) depend upon:

2) Covalent modification (e.g., phosphorylation)

Enzyme kinetics

The reaction rates of some enzymes can be regulated by:

1) Allosteric modulation

Fig 3.10

Fig 3.11

AmanTypewritten Text-non-covalent bond to the enzyme

Energy metabolism encompasses the pathways needed to convert the energy in food (stored as fuel) to ATP to power cellular functions.

ATP

ADP + Pi

ATP consumption Movement Membrane transport Molecular synthesis

ATP production Carbohydrates Lipids (fats) Proteins

Two general processes, involving enzyme catalyzed reactions, support ATP production and maintain cellular [ATP] homeostasis:

a) Substratelevel phosphorylation (occurs in absence of O2, such as anaerobic glycolysis)

b) Oxidative phosphorylation (depends on supply of O2 for oxidation reactions in mitochondria. The primary mode of ATP production in most cells)

Energy Metabolism

HEAT

HEAT

Glucose oxidation

Glucose oxidation is the central reaction of energy metabolism:

Fig 3.22

a) Glycolysis b) Linking step c) Krebs cycle d) Oxidative

phosphorylation (OxPhos)

Glycolysis breaks down glucose and produces two molecules of pyruvate, and occurs in the cytosol

Glycolysis and linking step Fig 3.15

Pyruvate enters the mitochondrion, where the linking step converts it to acetyl-coA

Fig 3.16

These steps result in acetyl-coA for the Krebs cycle, NADH to enter OxPhos, and ATP

AmanTypewritten Textpyruvate dehydrogenase is phosphorylated by an enzyme that regulates itself -when phosphorylated, the enzyme is inactivated, vital enzyme required for the oxidative phosphorylation

Fig 3.14

For reference, do not memorize

Glycolysis

Glycolysis is a pathway with several steps, each catalyzed by a different enzyme

(10 glycolytic enzymes are involved in glucose is oxidation)

AmanTypewritten Text-do not memorize the steps

Acetyl-coA enters the Krebs cycle in the mitochondrial matrix

Krebs cycle (or Tricarboxylic acid cycle)

Fig 3.18

This results in more ATP, as well as more NADH and FADH2 to enter OxPhos. NADH and FADH2 are termed reducing equivalents because they act as temporary electron carriers

AmanTypewritten Text-in the Krebs cycle, Acetyl-CoA is broken down to produce ATP, NADH and FADH2 (reducing equivalents, since they are electron carriers)

Krebs cycle (or Tricarboxylic acid cycle)

Fig 3.17

For reference,

do not memorize

Eight enzymes are involved in the Krebs cycle

AmanTypewritten Text-do not memorize

NADH and FADH2 donate their electrons to electron acceptors in the electron transport chain, and are oxidized to NAD+ and FAD

Oxidative phosphorylation

Fig 3.20

Electrons move through the electron transport system until they reduce O2 to H2O, and the energy released during this process is used to pump protons out of the mitochondrial matrix

Protons then move back into the matrix (down their concentration gradient) through the ATP synthase to make ATP

Electron transport chain

Fig 3.19

For reference, do not memorize

There are several electron transfer steps, involving several enzymes and coenzymes

Glucose oxidation

Fig 3.22

Complete oxidation of glucose yields ~34 ATP, most of which come from OxPhos In the absence of oxygen, glycolysis is followed by lactate production, yielding only 2 ATP

Fig 3.23

AmanTypewritten Text-one glucose molecule renders 38 ATP (34 of which is from oxidative phosphorylation)

Diversity of fuel oxidation

Fig 3.24

The particular fuel used, and its rate of oxidation, can be regulated depending on the needs of the organism

Carbohydrates (glycogen, glucose, and other sugars), lipids, and proteins can all be metabolized to make ATP by oxidative phosphorylation

ATP yield is greatest when oxidative phosphorylation is efficient, with perfect coupling between proton pumping and ATP synthesis

Uncoupling

AmanTypewritten Text-oxidative phosphorylation is efficient when all the protons are being pumped from the intermembrane space through ATP synthase to produce ATP -however, some protons leak through the membrane (renders inefficient oxidative phosphorylation)-uncoupling proteins, the excess leaked proteins are converted into heat (ex. brown fat in babies)

AmanTypewritten Text-mice, uncoupling protein remains throughout their lifetime

ATP yield is greatest when oxidative phosphorylation is efficient, with perfect coupling between proton pumping and ATP synthesis

Uncoupling

Animal Physiology Fig 8.4

Key to intracellular homeostasis, membrane transport is one of the primary ATP demanding processes in the cell

The properties and composition (lipids, transport proteins, etc.) of the cell membrane are key to maintaining cellular homeostasis

Membrane transport Chapter 4

Fig 3.13

Fuel oxidation

AmanTypewritten Text-membrane transport uses the most ATP

AmanSticky Note

Membranes 1) Very important as selective barriers

2) Transport proteins dictate movement in and out of cells or organelles

3) Membrane proteins can also be involved in detecting chemical messengers at the cell surface

Fig 2.16

Transport processes include: 1. Passive transport:

Mechanisms that DO NOT require ATP a) Simple diffusion through lipid

bilayer b) Carrier-mediated diffusion

through transmembrane protein channel

2. Active transport: Mechanisms that DO require ATP and dedicated transport proteins c) Primary active transport d) Secondary active transport (we will define these later)

Transport across membranes is very important for maintaining intracellular and extracellular homeostasis

Membrane transport

AmanTypewritten Text-do not memorize

Simple diffusion Diffusion: the movement of molecules from one location to another due to random thermal motion Concentration gradients provide a chemical driving force for diffusion, driving the movement solute from regions of higher to lower concentration until uniformly distributed

Flux: rate of solute movement per unit time Fig 4.1

Flux 1 Flux 2

Molecules diffuse in all directions, but the net flux (= flux 1- flux 2) is in the direction of lower concentration Gradient for diffusion causes downhill movement of solute overall. Net flux is zero at diffusive equilibrium

Simple diffusion

Example 2 Diffusion of solute across a barrier

Example 1 Diffusion of dye in water over time

Diffusion times (t) are proportional to distance2 (x2) over which diffusion occurs. Diffusion is only effective over short distances.

Fig 4.6

Simple diffusion

Net diffusion flux rate is proportional to the concentration difference between two locations (C)

Fig 4.7

Net diffusion flux rate across a membrane is also proportional to membrane permeability

(describes the ease of passage of a substance across a membrane)

Fig 4.9

AmanTypewritten Text-membranes that are more permeable allow for efficient diffusion (greater net flux)

Ficks Law

Net diffusion flux rate across a membrane (F) based on chemical driving forces alone follows Ficks Law

F= KpA ([X]o[X]i)

Netuxrate

Membranearea

Permeabilityconstant

Concentra/ondierence

acrossmembrane

Kptakesintoaccountmembranethickness(thinnermembraneshavepermeability)

ii) Solubility in lipid bilayers (nonpolar vs polar)

iii) Size and shape of the molecule (e.g., smaller molecules often diffuse faster)

i) Temperature (diffusion generally increases with temperature)

O2, CO2, fatty acids and steroid hormones are nonpolar and diffuse more rapidly than charged/polar solutes (i.e. most organic compounds)

The permeability constant is affected by:

AmanTypewritten Text-charged molecules cannot diffuse across membranes

Electrical driving forces for diffusion Net diffusion flux rate of a charged solute (ion) across a membrane also depends upon electrical driving forces Fig 4.3

The magnitude of Vm and the valency (charge) of the ion dictates the electrical driving force

Fig 4.4

There is a voltage, or membrane potential (Vm), across the cell membrane (typically -50 to -100 mV inside), due to maintenance of distinct ionic composition inside and outside cells

AmanTypewritten Text-negative charge inside the cell and positive charge outside the cell, a positive molecule will be diffused into the cell and a negative molecule will be diffused out of the cell

Electrochemical driving forces for diffusion

The electrochemical driving force, the combined effect of chemical and electrical forces, dictates the diffusion of ions across membranes

The equilibrium potential (Ek) for an ion is the Vm at which the electrical and chemical driving forces are equal (a)

Fig 4.5

When Vm is not equal to the Ek for a particular ion, there is an electrochemical driving force tending to cause diffusion (b,c)

AmanTypewritten Text-due to the chemical driving force potassium will be driven out of the cell as there is more potassium inside the cell -due to the electrical driving force, potassium will be diffused into the cell as there is a negative charge in the cell

AmanTypewritten Text-the chemical driving force and the electrical driving force balance out (equilibrium potential)

AmanTypewritten Text-if the voltage increases, the electrical driving force decreases, allowing more potassium molecules to be diffused out -if the voltage decreases, the electrical driving force is stronger

Nernst equation

The equilibrium potential differs between ions, depending on (i) an ions concentration inside and outside the cell and (ii) its valency

Ek can be calculated with the Nernst equation

Ek=lnRT [ion]outside

Faradayconstant

Temperature(inK)

Idealgasconstant

Valency(alwaysaposi/venumber)

[ion]insideFzRa/oofion

concentra/ons

Question: [K+] is much higher inside cells while [Na+] is much higher outside cells. Which ion has a positive EK?

We will discuss electrochemical gradients in more detail when we discuss neurophysiology

AmanTypewritten Text-

AmanTypewritten Text-do not memorize

AmanTypewritten Text-sodium, if the [ion]outside term is large, the Ek term is positive

Carrier-mediated diffusion

Fig 4.11

Carrier-mediated diffusion (or facilitated diffusion) can occur through protein channels/carriers, often for substances with otherwise low membrane permeability (e.g., Na+ channel at right)

The rate of facilitated diffusion is dictated by many of the same factors as simple diffusion, except that the rate is saturable (when a channels maximum capacity is reached)

Fig 4.12

Channels are selective for particular ions (based on size, charge, etc.), and can be regulated open/closed

AmanTypewritten Text-as the concentration gradient increases the net flux stabilizes due to the fact that the channel's maximum capacity is reached

Primary active transport

Fig 4.11

Membrane proteins that carry out primary active transport must hydrolyze ATP directly to harness energy, so they can transport ions against their electrochemical gradients

Na+/K+-ATPase (or Na+/K+ pump) is present in nearly all cells, sets intracellular & extracellular [K+] & [Na+], and establishes Vm

Frequently called ATPases and/or ion pumps

AmanTypewritten Text-without active transport, the concentration of ions would always be the same outside the cell and inside the cell

Primary active transport must constantly counteract passive ion leak across membranes to maintain the distinct ionic composition in the intracellular and extracellular environments

Leak is regulated by channels, in a manner that differs between ions (well discuss why later)

Primary active transport

Fig 4.16

Ion pumping is one of the most ATP demanding processes in cells

AmanTypewritten Text-primary active transport removes Na+ from cells when Na+ leaks into cells

Secondary active transport

For example, many transporters use the [Na+] gradient established by Na+/K+-ATPase to transport other substances against their electrochemical gradient

Transporters involved in secondary active transport can be co-transporters or counter-transporters (also called antiporters)

Secondary active transport is carried out by proteins that do not themselves hydrolyze ATP, but it relies upon ionic gradients established by other ATPases

Na+/organic substrate cotransporter

Fig 4.15

AmanTypewritten Text-various transporters diffuse other substrates with Na+ against its concentration gradients

AmanTypewritten Text-the channel itself is not an ATPase, but relies on pre-established concentration gradients to diffuse other molecules against its concentration gradient

1. Passive transport: Transport DOWN a concentration gradient, and therefore DOES NOT require ATP a) Simple diffusion b) Facilitated diffusion

2. Active transport: Transport AGAINST a concentration gradient, and DOES require ATP c) Primary active transport d) Secondary active transport (e.g., glucose uptake against its concentration gradient by co-transport with Na+ relies on the Na+ gradient established by Na+/K+-ATPase)

Categorized based on the process, and not the nature of the individual proteins involved

Summary of transport processes

Summary of transport processes

Membrane transport is one of the most ATP demanding processes in the cell

Channel arrest

AmanTypewritten Text-turtles bury themselves in muck in the winter, do not breathe -use lactic fermentation to minimize use of the mitochondria-shuts down the Na/K+ channels to minimize ATP need

Membrane transport is one of the most ATP demanding processes in the cell

Channel arrest

Normally, some ion leak occurs through membrane channels, counteracted by Na/K-ATPase

Summer active turtle

O2

Winter turtle without O2

41

Osmosis Osmosis is the passive diffusion of water across membranes. Membranes are permeable to water, due to its small size, even though it is a polar molecule

Water diffusion occurs down its concentration gradient. Water concentration depends on the osmolarity (total solute particle concentration) of the solution

The higher the osmolarity the lower the water concentration, so osmosis occurs in the direction of higher osmolarity

1 mole of dissolved particles = 1 osmolar solution.

e.g., 1M of glucose in solution = 1 osmole e.g., 1M of NaCl = 2 osmoles since it ionizes in solution to Na+ & Cl-

Fig 4.17

Tonicity While osmolarity refers to the total solute concentration, tonicity is a function of the concentration of non-permeating solutes (those unable to cross the cell membrane) outside relative to inside the cell The tonicity of a solution dictates the behaviour of a cell in that solution

Isotonic = equal concentration of non-permeating solute outside (300 mOsm) and inside (300 mOsm) cell Hypertonic = higher concentration of non-permeating solute outside (400 mOsm) than inside (300 mOsm) Hypotonic = lower concentration of non-permeating solute outside (200 mOsm) than inside (300 mOsm)

Nochangeincellvolume

300mOsm

Cellshrinks400mOsm

Cellswells

200mOsm

Tonicity

Over time

Fig 4.19

What happens when a cell containing 300 mOsm of non-permeating solute is placed into a solution containing 300 mOsm of permeating solute (urea)?

The permeating solute will enter the cell, diffusing down its concentration gradient, but the non-permeating cell will not. This creates the osmotic driving force for water diffusion into the cell hypotonic solution

Tonicity

Fig 4.20

Cell placed in a hypotonic solution:

Cell placed in a hypertonic solution:

Epithelial transport Transport of materials across entire cell layers

- The apical membrane faces external environment (lumen)

- The basolateral membrane faces the internal environment (interstitial fluid)

Different transport systems on the apical and basolateral membranes

Fig 4.23

Tight junctions form a selective barrier that limits movement between cells

Fig 2.27

Epithelial ion transport - example Epithelial transport of Na+ ions and glucose: Na+ and glucose enter the cell by a co-transporter across the apical membrane (secondary active transport), powered by the outward transport of Na+ across the basolateral membrane by the Na+/K+ pump (primary active transport) Buildup of intracellular glucose creates a driving force for its diffusion from the cell through basolateral channels

Fig 4.24

Epithelial water transport Epithelial water transport: movement of water across an epithelial cell layer occurs by osmosis, and depends upon the active transport of solutes to create an osmotic gradient

Left: pumps concentrate non-permeating solutes (dots) in the interstitial fluid (red arrows). Right: water diffuses across the epithelium, down its osmotic gradient (blue arrows).

Fig 4.25

Cell communication Chapter 5 Cell to cell communication is important for homeostasis Performed by intercellular chemical messengers 1) Hormones:

2) Neurotransmitters:

3) Autocrine / Paracrine agents:

48

- Local homeostatic responses, reach target cell by diffusion

- Auto = same cell; para = neighboring cells - e.g., nitric oxide, regulates blood vessels

- Secreted by nerve cell at synapse with target cell; Fast acting

- e.g., acetylcholine, controls heart rate

- Secreted by endocrine cells, they reach target cells via blood

- Slow acting - e.g., insulin, glucose homeostasis

Fig 5.2

Detectintercellularmessengersandconvertthemintoameaningfulintracellularresponse.Theyhavefourfeatures:

2)Amplica/on:

3)Desensi/za/on/adapta/on:

4)Integra/on:

Feedbackcanshutothereceptor

1receptorbindingcanleadto1,000,000intracellularsignals

response - +

Theintracellularsignalcanbetheresultofintegra/onofmul/plereceptorinputs

1) Specicity:Thesignalmolecule(S1)tsinitsreceptorwhileothers(S2)donot

Signal transduction pathways

Receptors The magnitude of a cells response is dictated by the number of receptors bound, which depends upon: 1) The messengers concentration 2) The number of receptors present 3) The receptors affinity for messenger

An increase in the number of receptors (R) increases the number bound by messenger

High affinity for messenger can increase # of bound receptors at the same [messenger]

Fig 5.10

1. Bind to lipophilic messengers (e.g. steroid hormones) 2. Act as transcription factors to alter gene transcription and

the translation of a specific protein 3. Receptors can be located in the cytosol or in the nucleus

Intracellular receptors

Fig 5.11

1) Channel-linked: (e.g., messenger binding opens ion channel)

Channel acts as receptor, called ligand-gated channel Respond very quickly to messenger binding

A change in the electrical properties of the cell can initiate the cellular response to messenger binding

Bind to lipophobic messengers, and there are 3 main types: 1. Channel-linked receptor 2. Enzyme-linked receptor 3. G-protein-linked receptor

Membrane-bound receptors

Fig 5.13

1) Channel-linked:

Fig 5.14

e.g., ligand-gated calcium channel

2)Enzyme-linked: Ligand-bindingdomainonextracellularsurfaceandanenzymeac/vesiteonintracellularside

Fig 5.15

Messenger binding alters the activity of the intracellular enzyme domain of the receptor

Tyrosine kinase receptors phosphorylate proteins to induce cellular responses (e.g., insulin receptor)

3)G-protein-linked: (Ac/vatemembraneproteinscalledG-proteinsthatbeginasignallingcascade)

a) Some G-protein-linked receptors regulate ion channels - These ion channels respond

more slowly to messenger binding, due to the time required for the alpha subunit to be activated and bind the channel

- In this case, the channel itself does not act as the receptor

Fig 5.16

3)G-protein-linked: (Ac/vatemembraneproteinscalledG-proteinsthatbeginasignallingcascade)

G-proteins can be stimulatory (Gs) or inhibitory (Gi)

a) Some G-protein-linked receptors regulate ion channels - These ion channels respond

more slowly to messenger binding, due to the time required for the alpha subunit to be activated and bind the channel

- In this case, the channel itself does not act as the receptor

Fig 5.17

b) Other G-protein-linked receptors regulate enzymes that produce second messengers (e.g., cyclic AMP, cAMP)

Second messengers - amplification Second messenger systems can amplify the signal from the intercellular messenger (first messenger) (e.g., G-protein receptor regulating cAMP production by adenylate cyclase; the type of receptor bound by adrenaline, the -adrenergic receptor)

Fig 5.19

Second messengers Chemical messengers of various types that we will discuss in the course make use of these various types of signal transduction mechanisms

Do not memorize, we will discuss some of these later