Exam3 Review II

1. GPCR; definition, function, example2. Second messengers3. Olive oil vs. blood pressure? PNAS Peres et al., 2008, 105, 13811.4. Insulin signaling5. Beta cell function6. Enzyme mechanisms7. Chorismate mutase8. Hemoglobin

GPCRs are 1. transmembrane receptors that sense molecules outside

the cell and activate inside signal transduction pathways.

2. What is wrong with this definition? 5P

Buck, L. and Axel, R. (1991) Cell, vol. 65, 175-187.

The most common second messengers

• Ca2+ calcium is the most common!• IP3 inositol triphosphate• DAG diacylglycerol• NO· nitric oxide• cAMP cyclic AMP

cAMP cycle: GPCR->Gs->adenylyl cyclase->cAMP

Cyclic AMP phosphodiesterase breaks down cAMP to 5’-AMP

2P

2P

How Oleic Acid in Olive Oil Reduces Blood Pressure?

(take home question, no points)

VASCULAR SMOOTH MUSCLE

AM

P c

GM

P c

PKA

PKG

MUSCULARRELAXATION

VASODILATION CROSS-TALK

Endothelium-dependent

REGULATION OF BLOOD PRESSURE

AC

AM

P c

PKA

G proteins

Q. Why is diabetes related to obesity? 5P

Insulin Secretion

Crosstalk between tissuesMetabolic interchanges: Interconnected

P13K

FKHR

Akt

mTOR

PTEN

MEK 1/2

MAPK

BADGSK-3

SOS

Grb-2

Shc

Grb-2

SOS Ras

Raf

JunFOS Myc

p27Cyclin D-1

LigandLigand

SignalAdaptersand Enzymes

SignalCascade

EGFr dimer

MAPK = mitogen-activatedprotein kinase

P13k = phosphatidylinositol3-kinase

TranscriptionFactors

EGFR Activation and Signaling Pathways

Source: With permission from Amgen Inc.

Outline• What characteristic features define enzymes?• Can the rate of an enzyme-catalyzed reaction be defined in a

mathematical way?• What equations define the kinetics of enzyme-catalyzed

reactions?• What can be learned from the inhibition of enzyme activity?• What is the kinetic behavior of enzymes catalyzing bimolecular

reactions?• How can enzymes be so specific? • Are all enzymes proteins?• Is it possible to design an enzyme to catalyze any desired

reaction?

Catalysts Lower the Free Energy of Activation for a Reaction

Figure 13.5 Energy diagram for a chemical reaction (A→P) and the effects of (a) raising the temperature from T1 to T2, or (b) adding a catalyst.

Chemical Kinetics Provides a Foundation for Exploring Enzyme Kinetics

• Consider a reaction of overall stoichiometry as shown:

• The rate is proportional to the concentration of A

[ ] [ ]

[ ][ ]

A P

d P d Av

dt dtA

v k Adt

14.4 How Tightly Do Transition-State Analogs Bind to the Active Site?

Figure 14.5 The proline racemase reaction. Pyrrole-2-carboxylate and Δ-1-pyrroline-2-carboxylate mimic the planar transition state of the reaction.

Chorismate Mutase: A Model for Understanding Catalytic Power and Efficiency

Figure 14.28 The chorismate mutase reaction converts chorismate to prephenate in an intramolecular rearrangement.

The chorismate mutase reaction (and its uncatalyzed counterpart) occur via chair states

Figure 14.29 The critical H atoms are distinguished in this figure by blue and green colors.

A transition-state analog for the chair mechanism of chorismate mutase

Jeremy Knowles has shown that both the chorismate mutase and its uncatalyzed solution counterpart proceed via a chair mechanism. A transition state analog of this state has been characterized.

The Chorismate Mutase Mechanism

Figure 14.32 The carboxyvinyl group folds up and over the chorismate ring and the reaction proceeds via an internal rearrangement.

The Adenylyl Cyclase Reaction

Figure 15.18 The adenylyl cyclase reaction. The reaction is driven forward by subsequent hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase.

Hemoglobin

• Hemoglobin and myoglobin are oxygen transport and storage proteins

• Compare the oxygen binding curves for hemoglobin and myoglobin

• Myoglobin is monomeric; hemoglobin is tetrameric • Mb: 153 aa, 17,200 MW • Hb: two α chains of 141 residues, 2 β chains of 146

residues

Figure 15.20 O2-binding curves for hemoglobin and myoglobin

Fe2+ is coordinated by His F8

• Iron interacts with six ligands in Hb and Mb• Four of these are the N atoms of the porphyrin• A fifth ligand is donated by the imidazole side chain of

amino acid residue His F8• (This residue is on the sixth or “F” helix, and it is the 8th

residue in the helix, thus the name.)• When Mb or Hb bind oxygen, the O2 molecule adds to

the heme iron as the sixth ligand• The O2 molecule is tilted relative to a perpendicular to

the heme plane

Cooperative Binding of Oxygen Influences Hemoglobin Function

• Mb, an oxygen storage protein, has a greater affinity for oxygen at all oxygen pressures

• Hb is different – it must bind oxygen in lungs and release it in capillaries

• Hb becomes saturated with O2 in the lungs, where the partial pressure of O2 is about 100 torr

• In capillaries, pO2 is about 40 torr, and oxygen is released from Hb

• The binding of O2 to Hb is cooperative – binding of oxygen to the first subunit makes binding to the other subunits more favorable

Hemoglobin and Nitric Oxide• Nitric oxide (NO·) is a simple gaseous molecule

that acts as a neurotransmitter and as a second messenger in signal transduction (see Chapter 32)

• NO· is a high-affinity ligand for Hb, binding to the heme iron 10,000 times more tightly than O2

• So why is NO· not bound instantaneously to Hb, preventing its physiological effects?

• NO· reacts with the –SH of Cys93β, forming an S-nitroso derivative:

The Mechanism of Muscle Contraction is Based on Sliding Filaments

Figure 16.8 The sliding filament model of skeletal muscle contraction. The decrease in sarcomere length is due to decreases in the width of the I band and H zone, with no change in the width of the A band. The lengths of the thick and thin filaments do not change during contraction.

The Contraction Cycle: ATP Hydrolysis Drives Conformation Changes in Myosin

• Cross-bridge formation is followed by power stroke with ADP and Pi release

• ATP binding causes dissociation of myosin heads and reorientation of myosin head

• Details of the conformational change in the myosin heads are coming to light

• Evidence now exists for a movement of at least 35 Å in the conformation change between the ADP-bound state and ADP-free state

16.3 What Are the Molecular Motors That Orchestrate the Mechanochemistry of Microtubules?

Figure 16.15 (a) Rapid axonal transport along microtubules permits the exchange of material between the synaptic terminal and the body of the nerve cell. (b) Vesicles, multivesicular bodies, and mitochondria are carried through the axon by this mechanism.

• In axons, dyneins move organelles + to -, i.e., toward the nucleus • Kinesins move organelles - to +, i.e., away from the nucleus

ATP Binding and Hydrolysis Drive Hand-over-Hand Movement of Kinesin

Figure 16.17 A model for the motility cycle of kinesin. The two heads of the kinesin dimer work together to move processively along a microtubule.