KAPLAN_USMLE_STEP_1_LECTURE_NOTES_2018_BIOCHEMISTRY_and_GENETICS

Part I ● Biochemistry

Note

GTP is energetically equivalent to ATP:

GTP + ADP ↔GDP + ATP is catalyzed by nucleoside diphosphate kinase.

In the citric acid cycle, all the enzymes are in the matrix of the mitochondria except succinate dehydrogenase, which is in the inner membrane.

Key points:

•Isocitrate dehydrogenase, the major control enzyme, is inhibited by NADH and ATP and activated by ADP.

•α-ketoglutarate dehydrogenase, like pyruvate dehydrogenase, is a multienzyme complex. It requires thiamine, lipoic acid, CoA, FAD, and NAD. Lack of thiamine slows oxidation of acetyl-CoA in the citric acid cycle.

•Succinyl-CoA synthetase (succinate thiokinase) catalyzes a substratelevel phosphorylation of GDP to GTP.

•Succinate dehydrogenase is on the inner mitochondrial membrane, where it also functions as complex II of the electron transport chain.

•Citrate synthase condenses the incoming acetyl group with oxaloacetate to form citrate.

Glucose

Pyruvate Amino acids

PDH

Fatty acids

Acetyl-CoA Ketones (extrahepatic)

Alcohol

ELECTRON TRANSPORT CHAIN

AND OXIDATIVE PHOSPHORYLATION

The mitochondrial electron transport chain (ETC) carries out 2 reactions:

Although the value of ∆G should not be memorized, it does indicate the large amount of energy released by both reactions. The electron transport chain is a device to capture this energy in a form useful for doing work.

Sources of NADH, FADH2, and O2

Many enzymes in the mitochondria—including those of the citric acid cycle and pyruvate dehydrogenase—produce NADH, all of which can be oxidized in the electron transport chain and in the process, capture energy for ATP synthesis by oxidative phosphorylation.

•If NADH is produced in the cytoplasm, the malate or α-glycerol phosphate shuttle can transfer the electrons into the mitochondria for delivery to the ETC.

•Once NADH has been oxidized, the NAD can again be used by enzymes that require it.

FADH2 is produced by succinate dehydrogenase in the citric acid cycle and by the α-glycerol phosphate shuttle. Both enzymes are located in the inner membrane and can reoxidize FADH2 directly by transferring electrons into the ETC. Once FADH2 has been oxidized, the FAD can be made available once again for use by the enzyme.

O2 is delivered to tissues by hemoglobin. The majority of oxygen required in a tissue is consumed in the ETC. Its function is to accept electrons at the end of the chain, and the water formed is added to the cellular water.

Pathways

NADH NAD

O2 H2O

Delivered by hemoglobin

Figure I-13-2. Electron Transport

Chain

CYTOPLASM SIDE

(INTERMEMBRANE SPACE)

Barbiturates

– Rotenone (an insecticide)

Glycerol-P

Shuttle

FADH2

cyt C

– Oligomycin

ATP

ADP Pi

Uncouplers

2,4-DNP + H+ Aspirin (high doses) Thermogenin (brown adipose)

Capturing Chemical Energy as Electricity

The mitochondrial electron transport chain works like a chemical battery. In one location, an oxidation reaction is poised to release electrons at very high energy; in another location, a potential electron acceptor waits to be reduced. Because the 2 components are physically separated, nothing happens. Once the 2 terminals of the battery are connected by a wire, electrons flow from one compartment to the other through the wire, producing an electrical current or electricity. A light bulb or an electrical pump inserted into the circuit will run on the electricity generated. If no electrical device is in the circuit, all the energy is released as heat. The mitochondrial electron transport chain operates according to the same principle.

NADH is oxidized by NADH dehydrogenase (complex I), delivering its electrons into the chain and returning as NAD to enzymes that require it. The electrons are passed along a series of protein and lipid carriers that serve as the wire.

These include, in order:

•NADH dehydrogenase (complex I) accepts electrons from NADH

•Coenzyme Q (a lipid)

•Cytochrome b/c1 (an Fe/heme protein; complex III)

•Cytochrome c (an Fe/heme protein)

•Cytochrome a/a3 (a Cu/heme protein; cytochrome oxidase, complex IV) transfers electrons to oxygen

All these components are in the inner membrane of the mitochondria as shown below. Succinate dehydrogenase and the α-glycerol phosphate shuttle enzymes reoxidize their FADH2 and pass electrons directly to CoQ.

Proton Gradient

The electricity generated by the ETC is used to run proton pumps (translocators), which drive protons from the matrix space across the inner membrane into the intermembrane space, creating a small proton (or pH) gradient. This is similar to pumping any ion, such as Na+, across a membrane to create a gradient. The 3 major complexes I, III, and IV (NADH dehydrogenase, cytochrome b/c1, and cytochrome a/a3) all translocate protons in this way as the electricity passes through them.

The end result is that a proton gradient is normally maintained across the mitochondrial inner membrane. If proton channels open, the protons run back into the matrix. Such proton channels are part of the oxidative phosphorylation complex.

Matrix

Figure I-13-4. Mitochondrion

ATP synthesis by oxidative phosphorylation uses the energy of the proton gradient and is carried out by the F0F1 ATP synthase complex, which spans the inner membrane. As protons flow into the mitochondria through the F0 component, their energy is used by the F1 component (ATP synthase) to phosphorylate ADP using Pi.

Bridge to Pathology

A genetic defect in oxidative phosphorylation is one cause of Leigh syndrome, a rare neurological disorder.

Part I ● Biochemistry

Bridge to Pathology

Ischemic Chest Pain

Patients with chest pain whose symptoms are suggestive of acute myocardial infarction (AMI) are evaluated by electrocardiogram and serial cardiac enzymes. Although myocardial-specific CK-MB has been used as an early indicator of an AMI, troponin levels are rapidly replacing it.

Troponin I and troponin T are sensitive and specific markers that appear 3–6 hours after the onset of symptoms, peak by 16 hours, and remain elevated for nearly a week. In the absence of ST-segment elevation on the EKG, elevated troponin I and troponin T are useful indicators for those at high risk for evolving myocardial infarction. LDH isozyme analysis may be helpful if a patient reports chest pain that occurred several days previously because this change (LDH1 > LDH2) peaks 2–3 days following an AMI.

On average, when an NADH is oxidized in the ETC, sufficient energy is contributed to the proton gradient for the phosphorylation of 3 ATP by F0F1 ATP synthase. FADH2 oxidation provides enough energy for approximately 2 ATP. These figures are referred to as the P/O ratios.

Hypoxia deprives the ETC of sufficient oxygen, decreasing the rate of ETC and ATP production. When ATP levels fall, glycolysis increases and, in the absence of oxygen, will produce lactate (lactic acidosis). Anaerobic glycolysis is not able to meet the demand of most tissues for ATP, especially in highly aerobic tissues like nerves and cardiac muscle.

In a myocardial infarction (MI), myocytes swell as the membrane potential collapses and the cell gets leaky. Enzymes are released from the damaged tissue, and lactic acidosis contributes to protein precipitation and coagulation necrosis.

The ETC is coupled to oxidative phosphorylation so that their activities rise and fall together. Inhibitors of any step effectively inhibit the whole coupled process, resulting in:

•Decreased oxygen consumption

•Increased intracellular NADH/NAD and FADH2/FAD ratios

•Decreased ATP

Important inhibitors include cyanide and carbon monoxide.

Cyanide is a deadly poison because it binds irreversibly to cytochrome a/a3, preventing electron transfer to oxygen, and producing many of the same changes seen in tissue hypoxia. Sources of cyanide include:

•Burning polyurethane (foam stuffing in furniture and mattresses)

•Byproduct of nitroprusside (released slowly; thiosulfate can be used to destroy the cyanide)

Nitrites may be used as an antidote for cyanide poisoning if given rapidly. They convert hemoglobin to methemoglobin, which binds cyanide in the blood before reaching the tissues. Oxygen is also given, if possible.

Carbon monoxide binds to cytochrome a/a3 but less tightly than cyanide. It also binds to hemoglobin, displacing oxygen. Symptoms include headache, nausea, tachycardia, and tachypnea. Lips and cheeks turn a cherry-red color. Respiratory depression and coma result in death if not treated by giving oxygen. Sources of carbon monoxide include:

•Propane heaters and gas grills

•Vehicle exhaust

•Tobacco smoke

•House fires

•Methylene chloride–based paint strippers

Other inhibitors include antimycin (cytochrome b/c1), doxorubicin (CoQ), and oligomycin (F0).

Uncouplers are chemicals that decrease the proton gradient, causing:

•Decreased ATP synthesis

•Increased oxygen consumption

•Increased oxidation of NADH

Because the rate of the ETC increases, with no ATP synthesis, energy is released as heat. Important uncouplers include 2,4-dinitrophenol (2,4-DNP) and aspirin (and other salicylates). Brown adipose tissue contains a natural uncoupling protein (UCP, formerly called thermogenin), which allows energy loss as heat to maintain a basal temperature around the kidneys, neck, breastplate, and scapulae in newborns.

Recall Question

Which of the following substrates is used in heme synthesis?

A.Citrate

B.Fumarate

C.Succinate

D.Succinyl-CoA

Answer: D

When molecular oxygen (O2) is partially reduced, unstable products called reactive oxygen species (ROS) are formed. These react rapidly with lipids to cause peroxidation, with proteins, and with other substrates, resulting in denaturation and precipitation in tissues. Reactive oxygen species include:

•Superoxide (O2·-)

•Hydrogen peroxide (H2O2)

•Hydroxyl radical (OH.)

The polymorphonuclear neutrophil produces these substances to kill bacteria in the protective space of the phagolysosome during the oxidative burst accompanying phagocytosis. Production of these same ROS can occur at a slower rate wherever there is oxygen in high concentration. Small quantities of ROS are inevitable by-products of the electron transport chain in mitochondria. These small quantities are normally destroyed by protective enzymes such as catalase. The rate of ROS production can increase dramatically under certain conditions, such as reperfusion injury in a tissue that has been temporarily deprived of oxygen. ATP levels will be low and NADH levels high in a tissue deprived of oxygen (as in an MI). When oxygen is suddenly introduced, there is a burst of activity in the ETC, generating incompletely reduced ROS.

Bridge to Pharmacology

Aspirin in high doses used to treat rheumatoid arthritis can result in uncoupling of oxidative phosphorylation, increased oxygen consumption, depletion of hepatic glycogen, and the pyretic effect of toxic doses of salicylate. Depending on the degree of salicylate intoxication, symptoms can vary from tinnitus to pronounced CNS and acid-base disturbance.

Part I ● Biochemistry

Bridge to Medical Genetics

Mitochondrial Diseases

•Leber hereditary optic neuropathy

•Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS)

•Myoclonic epilepsy with ragged-red muscle fibers

Defenses against ROS accumulation are particularly important in highly aerobic tissues and include superoxide dismutase and catalase. In the special case of erythrocytes, large amounts of superoxide are generated by the spontaneous dissociation of the oxygen from hemoglobin (occurrence is 0.5–3% of the total hemoglobin per day). The products are methemoglobin and superoxide. The processes that adequately detoxify the superoxide require a variety of enzymes and compounds, including superoxide dismutase, catalase, as well as glutathione peroxidase, vitamin E in membranes, and vitamin C in the cytoplasm. Low levels of any of these detoxifying substances result in hemolysis. For example, inadequate production of NADPH in glucose 6-phosphate dehydrogenase deficiency results in accumulation of the destructive hydrogen peroxide (Chapter 14).

The circular mitochondrial chromosome encodes 13 of the >80 proteins that comprise the major complexes of oxidative phosphorylation, as well as 22 tRNAs and 2 rRNAs. Mutations in these genes affect highly aerobic tissues (nerves, muscle), and the diseases exhibit characteristic mitochondrial pedigrees (maternal inheritance).

Key characteristics of most mitochondrial DNA (mtDNA) diseases are lactic acidosis and massive proliferation of mitochondria in muscle, resulting in ragged red fibers. Examples of mtDNA diseases are:

•Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS)

•Leber hereditary optic neuropathy

•Ragged-red muscle fiber disease

Coordinate Regulation of the Citric Acid Cycle and Oxidative Phosphorylation

The rates of oxidative phosphorylation and the citric acid cycle are closely coordinated, and are dependent mainly on the availability of O2 and ADP.

•If O2 is limited, the rate of oxidative phosphorylation decreases, and the concentrations of NADH and FADH2 increase. The accumulation of NADH, in turn, inhibits the citric acid cycle. The coordinated regulation of these pathways is known as “respiratory control.”

•If O2 is adequate, the rate of oxidative phosphorylation depends on the availability of ADP. The concentrations of ADP and ATP are reciprocally related; an accumulation of ADP is accompanied by a decrease in ATP and the amount of energy available to the cell.

–Therefore, ADP accumulation signals the need for ATP synthesis.

–ADP allosterically activates isocitrate dehydrogenase, thereby increasing the rate of the citric acid cycle and the production of NADH and FADH2. Elevated levels of these reduced coenzymes, in turn, increase the rate of electron transport and ATP synthesis.