Biochem 1 Acidic dissociation

Biochem 2 1. General expression: HA is the acid (proton donor) and

A- is the conjugate base (proton acceptor): 2. An acid dissociates in water to yield a hydrogen ion (H+) and

its conjugate base 3. A base combines with H+ in water to form its conjugate acid

k1[HA] = forward rate, k-1[H+][A-] = reverse rate

Acid (acetic acid) Conjugate base (acetate) CH3COOH --> H+ + CH3COO-

Ammonia (base) Ammonium ion (conjugate acid) NH3 + H+ --> NH4+

k1 HA --> H+ + A- k-1

Biochem 3 Measures of acidity

Biochem 4 1. pKa

When forward & reverse rates are equal, acidic dissociation constant, Ka, is defined by: o k1/k-1 = [H+][A-] / [HA] = Ka

Expresses the STRENGTH OF AN ACID pKa = -log[Ka] Strong acid has pKa of 2 (H+ binds loosely to conjugate base) Weak acid has a pKa of 10 (H+ binds tightly to conjugate base)

2. pH Henderson-Hasselbalch equation: pH = pKa + log [A-]/[HA] A measure of the ACIDITY OF A SOLUTION pH = -log[H+] Neutral solution has a [H+] of 10-7 ! pH = 7 Acidic solution has a [H+] > 10-7 ! pH < 7 Alkaline solution has a [H+] < 10-7 ! pH > 7

Biochem 5 Buffers and Buffering capacity

Biochem 6 1. A solution that contains a mixture of a weak acid

and its conjugate base 2. It resists changes in [H+] on addition of acid or alkali 3. The buffering capacity of a solution is determined by the

acid-base concentration and the pKa of the weak acid Maximum buffering effect occurs when:

o [weak acid] = [conjugate base] When the buffer effect is at its maximum:

o pH of the solution = pKa of the acid 4. Buffering effect is seen on a titration curve for a weak acid

The shape of the curve is the same for all weak acids At the midpoint of the curve, the pH = pKa The buffering region extends one pH unit above and below

the pKa

Biochem 7 What acid-base pair is an effective buffer in physiologic fluids? What acid-base pair is the principal buffer in plasma and extracellular fluid (ECF)?

Biochem 8 1. What acid-base pair is effective buffer in physiolog fluids?

H2PO4- and HPO42- 2. What acid-base pair is the principal buffer in plasma and ECF?

CO2-H2CO3-HCO3- system (carbon dioxide-carbonic acid-bicarb) CO2 + H2O carbonic anhydrase! H2CO3 "! H+ + HCO3- Note: carbonic anhydrase converts CO2 to H2CO3 in RBCs In this system, CO2 is an acid, so H-H equation is:

o pH = 6.1 + log [HCO3-] / (0.0301)PCO2 This system is effective around physiologic pH of 7.4, even though

the pKa is only 6.1, for 4 reasons: o Supply of CO2 from oxidative metabolism is unlimited, so effective

concentration of CO2 is very high o Equilibration of CO2 with H2CO3 is very rapid o CO2 removal by lungs allows for rapid changes in [H2CO3] o Kidney can retain or excrete HCO3-, thus changing the concentration

of the conjugate base

Biochem 9 Acid-Base Disorders: Acidosis and Alkalosis

Biochem 10 1. Acidosis

Occurs when pH of blood and ECF falls < 7.35 Results in CNS depression When severe, can lead to coma and death Respiratory acidosis: pCO2 as a result of hypoventilation Metabolic acidosis: [HCO3-] as a result of the addition of

an acid stronger than H2CO3 to the ECF 2. Alkalosis

Occurs when pH of blood and ECF is >7.45 Leads to neuromuscular hyperexcitability When severe, can result in tetany Respiratory alkalosis: pCO2 as result of hyperventilation Metabolic alkalosis: [HCO3-] as a result of excess acid

loss (e.g., vomiting) or addition of a base (e.g., oral antacids)

Biochem 11 Diabetic ketoacidosis

Biochem 12 1. Combination of high blood levels of ketone bodies and a

metabolic acidosis 2. Pathogenesis

Uncontrolled insulin-dep DM (type 1) ! glucose utilization, hyperglycemia ! fatty acid oxidation

fatty acid oxidation ! excessive production of acetoacetic and 3-hydroxybutyric acids and acetone (ketone bodies)

Acids dissociate at body pH and release H+ ! metabolic acidosis 3. Clinical picture

Dehydration Lethargy

Vomiting Drowsiness

Coma

4. Therapy: correct the hyperglycemia, dehydration, & acidosis Insulin to correct the hyperglycemia Fluids (physiologic saline) to treat dehydration In severe cases: sodium bicarbonate to correct acidosis

Biochem 13 Amino acids grouped by the properties of their R-groups

Biochem 14 1. Aliphatic, nonpolar (hydrophobic)

Glycine Alanine

Valine Leucine

Isoleucine Proline

2. Aromatic, nonpolar Phenylalanine Tyrosine Tryptophan

3. Sulfer-containing Cysteine Methionine

4. Hydroxyl, mildly polar (uncharged, hydrophilic) Serine Threonine

5. Basic, polar Lysine Arginine Histidine

6. Acidic, polar Aspartic acid Asparagine

Glutamic acid Glutamine

Pg. 7 for structures

Biochem 15 Secondary structures of proteins and collagen

Biochem 16 1. Secondary structure = arrangement of H bonds between

peptide nitrogens & carbonyl oxygens of different amino acids 2. Helical coils

Hydrogen-bonded nitrogens & oxygens are on nearby amino acids Right-handed alpha helix most common

o Alpha-keratin in hair and nails o Myoglobin has several alpha-helical regions

Proline, glycine, and asparagine helix breakers 3. Beta sheets (pleated sheets) may run parallel or antiparallel

Hydrogen bonds between residues on neighboring peptide chains 4. Left-handed helical strands

Wound into a supercoiled triple helix in collagen Collagen major structural protein in the body

o Primary structure: repeating glycine-X-Y sequences o X and Y are freqeuntly proline or lysine o Contains hydroxyproline & hydroxylysine

Biochem 17 Protein Denaturation Agents

Biochem 18 1. Extremes of pH (e.g., strong acid or alkali) 2. Ionic detergents (e.g., sodium dodecylsulfate/SDS) 3. Chaotropic agents (e.g., urea, guanidine) 4. Heavy metal ions (e.g., Hg++) 5. Organic solvents (e.g., alcohol or acetone) 6. High temperature 7. Surface films (e.g., as when egg whites are beaten)

Biochem 19 Sickle cell anemia

Biochem 20 1. Caused by mutant sickle cell hemoglobin (Hgb S)

Hydrophobic valine replaces hydrophilic glutamate at position 6 of the beta-chain of normal hemoglobin A (Hgb A)

2. Sickle cell disease Individuals with homozygous genotype (SS) Have only Hgb S in their RBCs Symptoms

o Severe anemia: deoxy Hgb S produces fibrous precipitates ! formation of sickle cells ! shorter life span ! severe anemia

o Acute episodes of vaso-occlusion sickle cell crisis Sickle cells cant pass thru capillaries ! vasocclusion Disabling pain that requires hospitalization

3. Sickle cell trait Individuals with heterozygous genotype (AS) Have both Hgb A and Hgb S in their RBCs Symptoms

o Usually asymptomatic o Episodes of hematuria

Biochem 21 Scurvy

Biochem 22 1. Defective collagen synthesis resulting from a

vitamin C (ascorbic acid) deficiency 2. Consequences of abnormal collagen

Defective wound healing Defective tooth formation Loosening of teeth

Bleeding gums Rupture of capillaries

3. Ascorbic acid is required for hydroxylation of proline and lysine during post-translational modification of collagen

4. Pathogenesis Hydroxylating rxn requires hydroxylase, O2, & Fe2+ Vit C is required to maintain iron in its oxidation state (Fe2+) Hydroxyproline forms H-bonds that stabilize collagen helix Symptoms of scurvy are thus the result of weakend collagen

when these hydrogen bonds are missing

Biochem 23 Free energy change

Biochem 24 1. The quanitity of energy from chemical reactions

that is available to do work (G) 2. The G of a rxn A + B "! C + D is:

G = G0 + RTln ([C][D]) / [A][B]) o where G0 is the standard free-energy change

(when concentrations of all reactants and products are 1M and pH = 7), R is the gas constant (1.987 cal/molK) and T is the absolute temperature

3. When the rxn has reached equilibrium: G0 = RTlnKeq

Biochem 25 Thermodynamic spontaneity: Exergonic and Endergonic Rxns

Biochem 26 1. Exergonic rxns are spontaneous (rxn goes to the right)

Keq > 1 G0 is negative Final concentration of the products, C and D, is greater than

that of the reactants, A and B 2. Endergonic rxns are nonspontaneous (rxn goes to the left)

Keq < 1 G0 is positive Final concentration of the reactants, A and B, is greater than

that of the products, C and D 3. Note: G0 CANNOT predict spontaneity under intracellular

conditions Intracellular spontaneity is a function of actual

concentrations as well as Keq, G, NOT G0

Biochem 27 Enthalpy, entropy, and free-energy change

Biochem 28 1. Enthalpy (H)

The amount of heat generated or absorbed in a rxn 2. Entropy (S)

Measure of the change in randomness or disorder of system when a salt crystal dissolves, when a solute diffuses from a

more concentrated to a less concentrated solution, and when a protein is denatured

when a complex molecule is synthesized from smaller substrates

3. Free-energy change (G) Is related to enthalpy and entropy as follows:

o G = H - T S (where T = absolute temp in Kelvins)

Biochem 29 Catalysts and the Rate of Reaction

Biochem 30 1. Rate of reaction

The G0 provides no info concerning the rate of conversion from A to B

When A is converted to B, it first goes through an energy barrier called the transition state, A-B

The activation energy (G) = energy required to scale the energy barrier and form the transition state

The greater the G, the lower the rate of the rxn converting A to B

2. Catalysts (mostly enzymes) result in a: Lower G Faster reaction rate

Biochem 31 Michaelis-Menten equation

Biochem 32 1. Describes the kinetics of enzyme rxns 2. In enzyme-catalyzed rxns:

k1 k3 E + S "! ES ! E + P k2

Where E=enzyme, S=substrate, P=product, k1-3 = rate constants 3. Velocity (v) of product formation is related to [ES]:

o v = k3[ES] where k3 is also called kcat 4. Michaelis-Menten eq predicts velocity if [enzyme] is constant:

Where Vm = max velocity & Km is the Michaelis constant 5. Km = [substrate] at which v = Vm ([S] = Km) 6. A plot of velocity versus [S] is a rectangular hyperbola

Vm[S] v = Km+[S]

Biochem 33 Lineweaver-Burk Equation

Biochem 34 1. Form of the Michaelis-Menten eq, which is

sometimes known as the double-reciprocal equation: 1 = Km + [S] = Km x 1 + 1 v = Vm[S] Vm [S] Vm

2. The slope is Km/Vm 3. The Y-intercept = 1/Vm 4. The X-intercept is 1/Km

Biochem 35 Enzyme Regulation: How doe Inhibitors affect the Lineweaver-Burk plots?

Biochem 36 1. Competitive inhibitors

apparent Km Vm remains the same slope X-intercept has smaller absolute value Y-intercept is unchanged

2. Noncompetitive inhibitors Vm Km unchanged slope X-intercept is unchanged Y-intercept is larger

3. Uncompetitive inhibitors (bind only to ES complex) Both Km & Vm are different ! lines on the plot are parallel

Biochem 37 Allosteric regulation of enyzmes: Definition, How do they affect Km, and Example of Hexokinase

Biochem 38 1. Low-molec wgt effector binds to enzyme at a specific

site other than active site (the allosteric site) & alters its activity 2. Allosteric enzymes usually have >1 subunit and >1 active site

Active sites that interact cooperatively: velocity vs, [S] = sigmoid Binding of 1 substrate facilitates binding of substrate at other sites

3. Effectors may have a + or effect on activity Positive effectors the apparent Km Negative effectors the apparaent Km

4. Example: muscle hexokinase Hexokinase catalyzes 1st rxn in use of glucose my muscle cells:

o Glucose + ATP ! glucose-6-P + ADP Hexokinase has a low Km compared to blood [glucose], so it is

saturated and operates at its Vm When glycolysis slows down, gluc-6-P accumulates ! gluc-6-P

allosterically inhibits hexokinase (keeps gluc-6-P in balance)

Biochem 39 Other mechanisms of enzyme regulation:

1. Induction/repression of enzyme synthesis 2. Covalent modificataion 3. Protein-protein interaction

Biochem 40 1. Induction/repression of enzyme synthesis

Cytochrome P450 enzymes in the liver (degrade and detoxify drugs) are induced by the drugs themselves

2. Covalent modificataion Phosphorylase (enzyme that breaks down glycogen) is

activated by phosphorylation of a specific hydroxyl group This phosphorylation is stimulated by hormones that elevate

blood glucose, such as glucagon and Epi 3. Protein-protein interaction between enzyme & regulatory

protein Pancreatic lipase (digests dietary fat) is assisted by colipase Colipase anchors lipase to the surface of fat droplets

Biochem 41 Mechanism and Treatment of Methanol & Ethylene glycol Poisoning

Biochem 42 1. Mechanism of poisoning

Toxicity is caused by the action of their metabolites In both cases, the 1st oxidation is carried out by alcohol

dehydrogenase o Methanol ! formaldehyde + formic acid

Eyes very sensitive to formaldehyde ! blindness o Ethylene glycol ! glycoaldehyde, oxalate, and lactate

Deposition of oxalate crystals in kidney ! kidney failure

2. Treatment Initial infusion of ethanol ! competitive substrate !

displaces methanol or ethylene glycol from active site of alcohol dehydrogenase

Prevents continued production of toxic metabolites

Biochem 43 Citric acid cycle: Location, Pathway, and Initial Substrate

Biochem 44 1. Location

Mitochondria (found in all cells except RBCs) 2. Pathway

It is the final common pathway of oxidiative metabolism

3. Initial Substrate: Acetyl Coenzyme A (ACETYL CoA) Condenses with oxaloacetate (OAA) to begin the cycle

4. Where does acetyl CoA come from? The catabolism of carbs, fats, & proteins

o Glucose catabolism eventually produces pyruvate ! acetyl CoA via pyruvate dehydrogenase

o Fatty acids generate acetyl CoA via -oxidation o Some amino acids are degraded to acetyl CoA

Biochem 45 What are the products of one revolution of the citric acid cycle?

Biochem 46 1. 2 CO2 (most CO2 from metabolism) 2. Regeneration of one mole of OAA 3. 3 NADH & 1 FADH2 ! 11 ATPs (via oxidative

phosphorylation) 4. 1 GTP ! 1 ATP TOTAL OF 12 ATPs/acetyl CoA

Oxidative Phosph: 1 NADH = 3 ATP 1 FADH2 = 2 ATP

Biochem 47 Describe the anaplerotic rxns that provide OAA and other citric acid cycle intermediates

Biochem 48 1. Pyruvate carboxylase in the liver & kidney:

Pyruvate + ATP + HCO3- "! OAA + ADP + Pi 2. Phosphoenolpyruvate (PEP) carboxykinase in heart and

skeletal muscle: PEP + CO2 + GDP "! OAA + GTP

3. Malic enzyme in many tissues: Pyruvate + HCO3- + NAD(P) "! Malate + NAD(P)+

4. Glutamate dehydrogenase in the liver: Glutamate + NAD(P)+ + H2O "! -ketoglutarate +

NAD(P)H + NH4+

Biochem 49 Regulation of the citric acid cycle

Biochem 50 1. Step: Acetyl CoA + OAA ! citrate

Enzyme: citrate synthase Inhibitors: ATP ( Km), long-chain acyl-CoA

2. Step: Isocitrate + NAD+ !-ketoglutarate + NADH + CO2 Enzyme: isocitrate dehydrogenase Allosteric activator: ADP Inhibitors: ATP, NADH

3. Step: -ketoglutarate + NAD+ + CoASH ! succinyl CoA + NADH + CO2 Enzyme: -ketoglutarate dehydrogenase (note: requires

same cofactors as the pyruvate dehydrogenase complex) Inhibitors: succinyl CoA, NADH

Regulated mainly by need for ATP, & therefore by supply of NAD+

Biochem 51 Electron Transport Chain (ETC) & Oxidative Phosphorylation (BOTH in MITOCHONDRIA)

Biochem 52

NADH! !Q! !cytochrome c ATP" ATP synthase-- Proton gradient"O2" H2O

NADH dehydrogenase

Ubiquinone-c oxidoreductase

(Cytochrome bc1)

Cytochrome oxidase

1. ETS: electrons pass from NADH or FADH2 to ultimately reduce O2 and produce H2O

2. Oxidative phosphorylation: uses energy derived from flow of electrons thru ETS to drive synthesis of ATP from ADP and Pi

NADH dehydrog. = Complex I, Succinate dehydrog. = Complex II (where FADH2 enters not pictured here), Cytochrome bc1 = Complex III, Cytochrome oxidase = Complex IV

Biochem 53 Chemiosmotic hypothesis

Biochem 54 1. Describes coupling of electron flow thru ETS to ATP 2. Respiratory complexes as proton pumps:

As electrons (e-) pass thru complexes I, III, & IV, hydrogens are pumped across inner membrane to intermembrane space

The [H+] in the intermembrane space relative to matrix This generates a proton-motive force as result of 2 factors:

o Difference in pH (pH) o Difference in electrical potential () between the

intermembrane space and the mitochondrial matrix 3. ATP synthase complex (complex V)

Hydrogen ions pass back into the matrix thru complex V, and in doing so, drive the synthesis of ATP o Passage of pair of e- from NADH to O2 ! 3ATP o Passage of e- pair from FADH2 to O2 (bypass I) !2ATP

Biochem 55 Uncouplers and Inhibitors of ETS

Biochem 56 1. Uncoupling

Carry H+ across inner mit membrane w/o going thru complex V This short-circuits the proton gradient and uncouples electron

flow from ATP synthesis Energy, instead of used to make ATP, is dissipated as heat Uncoupling agents:

o Dinitrophenol (2,4-DNP) former diet drug Caused blindness (retina has rate of oxidativ metblism)

o Thermogenin helps to maintain normal body temp Found normally in brown fat of newborn mammals

2. Inhibitors (via blocking e- flow thru complexes or direct action) Complex I Amobarbital (barbiturate), Rotenone (insecticide),

Piericidin A (antibiotic), Amytal Complex II Antimycin A (antibiotic) Complex IV Cyanide, Hydrogen sulfide, Carbon monoxide ATP synthase Oligomycin

Biochem 57 Carbohydrate digestion and absorption

Biochem 58 1. Digestion

Disacharides (sucrose),oligosacharides (dextrins),& polysacharides (starch) are cleaved into monosaccharides (glucose, fructose)

Starch: storage from of carbs in plants o Hydrolyzed to maltose, maltotriose, and -dextrins by -

amylase in saliva and pancreatic juice Disaccharides & oligosaccharides

o Hydrolyzed to monosaccharides by enzymes on the surface of epithelial cells in the small intestine

2. Absorption Monosaccharides absorbed directly by carrier-mediated transport These sugars (primarily glucose) travel via portal vein to liver for:

o Oxidation to CO2 and H2O for energy o Storage as glycogen o Conversion to triglyceride (fat) o Release into general circulation (as glucose)

Biochem 59 Glycogen metabolism

Biochem 60 [Glycogen: carb storage, found chiefly in liver & muscle]

1. Glycogenesis (glycogen synthesis) Activated substrate: Uridine diphosphate-glucose Glycogen synthase adds to nonreduc end of chains in -1,4 links Branching enzyme amylo (1!4) to (1!6) transglycosylase

creates branches w/-1,6 linkages Stimulator: insulin (via dephosphorylation in muscle, liver, & fat) Inhibitors: glucagon (liver), Epi (muscle & liver), phosphorylase

(liver), cAMP, Ca2+ (muscle) 2. Glycogenolysis (glycogen breakdown)

Phosphorylase releases units of glucose 1-P from nonreducing end Phosphoglucomutase converts glucose 1-P to glucose 6-P Debranching system releases glucose residues from -1,6 bonds Stimulators: sames as inhibitors of glycogenesis Inhibitor: insulin (via dephosphorylation in muscle, liver, and fat)

Biochem 61 Glycolysis: Location, Anaerobic and Aerobic

Biochem 62 1. Location: cytosol in most tissues of the body 2. Anaerobic (without oxygen)

Glucose ! 2Lactate + 2ATP Characteristic of skeletal muscle after prolonged

exercise Lactate dehydrogenase converts pyruvate to lactate

3. Aerobic: Glucose + 6O2 ! 6CO2 + 6H2O + 36-38 ATP Charactersitic of the brain NADH produced is oxidized by the mitochondrial ETS ATP is generated by oxidative phosphorylation

Biochem 63 Describe the first step in glycolysis

Biochem 64 1. Phosphorylation involves rxn of glucose in presence

of hexokinase OR glucokinase to form glucose 6-phosphate Hexokinase is found in the cytosol of most tissues:

o Low specificity (catalzyes phosphorylation of a wide variety of hexoses)

o Low Km (its saturated at normal blood [glucose]) o Inhibited by glucose 6-P (prevents cells from

accumulating too much glucose since phosphorylation traps glucose inside cells)

Glucokinase is present in the liver & pancreas (-cells): o High specificity for glucose o High Km (above the normal blood [glucose]) o Inhibited by fructose 6-P (ensures glucose will be

phosphorylated only as fast as it is metabolized)

Biochem 65 What happens in the 2 phases of glycolysis?

Biochem 66 1. In the first phase (5 reactions):

1 mole of glucose is converted to 2 moles of glyceraldehyde 3-P

2 moles of ATP are consumed for each mole of glucose 2. In the second phase (5 reactions):

Two moles of glyceraldehyde 3-P are oxidized to 2 moles of pyruvate

4 moles of ATP and 2 moles of NADH are generated for each mole of glucose

Biochem 67 What do the following do:

1. Glycerol phosphate shuttle 2. Malate-aspartate shuttle

Biochem 68 NADH produced in the cytosol DOES NOT pass through the mitochondrial inner membrane, but is instead shuttled in by: 1. Glycerol phosphate shuttle (most tissues)

Transfers electrons from cytosolic NADH to mitoch FADH2 It generates 2 ATP/cytosolic NADH = 36 moles of

ATP/glucose 2. Malate-aspartate shuttle (heart, muscle, & liver)

Transfers electrons to mitochondrial NADH It generates 3 ATP/cytosolic NADH = 38 ATP/glucose

Biochem 69 Gluconeogenesis

Biochem 70 1. Occurs primarily in the liver & kidney 2. Synthesis of glucose from small noncarb precursors (such as lactate and

alanine) 3. Involves the reversible rxns of glycolysis 4. To bypass nonreversible steps of glycolysis, separate rxns occur:

Conversion of pyruvate to PEP bypasses pyruvate kinase Conversion of fructose 1,6-bisphosphate to fructose 6-phosphate

by fructose 1,6-bisphosphatase bypasses phosphofructokinase Conversion of glucose 6-P to glucose by glucose 6-phosphatase

bypasses hexokinase 5. Glucose from gluconeogenesis is released into the bloodstream for

transport to tissues such as the brain and exercising muscle 6. Gluconeogenic substrates:

Lactate Pyruvate Glycerol Substances that can be converted to oxalacetate via the citric acid

cycle (such as amino acid carbon skeletons)

Biochem 71 Cori cycle

Biochem 72 1. Shuttling of gluconeogenic substrates between

RBCs and muscle to liver, allowing muscle to function anaerobically (net 2 ATP)

2. Lactate from exercising or ischemic muscle is carried by the circulation to the liver and serves as a substrate for gluconeogenesis

3. The liver releases the resynthesized glucose into the circulation for transport back to the muscle

Biochem 73 Regulation of glycolysis

Biochem 74 1. All are the irreversible steps:

Fructose 6-P ! fructose-1,6-BP via phosphofructokinase o Stimulators: AMP, fructose 2,6-BP (in liver) o Inhibitors: ATP, citrate o Rate-limiting step

D-glucose ! glucose-6-P via hexokinase/glucokinase* o Inhibitors: glucose-6-P

PEP ! pyruvate via pyruvate kinase o Inhibitors: ATP, alanine o Stimulators: fructose-1,6-BP (in muscle)

Pyruvate ! acetyl CoA via pyruvate dehydrogenase o Stimulators: CoA, NAD, ADP, pyruvate o Inhibitors: ATP, NADH, acetyl CoA

2. Induced by insulin

Biochem 75 Gluconeogenesis regulation

Biochem 76 1. All are the irreversible steps:

Pyruvate ! OAA via Pyruvate carboxylase (mitochond) o Requires biotin, ATP o Activated by acetyl CoA

OAA ! PEP via PEP carboxykinase o Requires GTP

Fructose-1,6-BP ! fructose-6-P via Fructose-1,6-BPase Glucose-6-P ! glucose via Glucose-6-phosphatase

2. These enzymes are only found in liver, kidney, intestinal epithel 3. Muscle cannot participate in gluconeogenesis 4. Hypoglycemia is caused by a deficiency of these key enzymes 5. Induced by glucocorticoids, glucagon, cAMP 6. Suppressed by insulin

Pnemonic: Pathway Produces Fresh Glucose

Biochem 77 Pyruvate dehydrogenase complex

Biochem 78 1. Contains 3 enzymes that require 5 cofactors:

Pyrophosphate (from thiamine) Lipoic acid CoA (from pantothenate) FAD (riboflavin) NAD (niacin)

2. Reaction: Pyruvate + NAD+ + CoA ! acetyl-CoA + CO2 + NADH

3. The complex is similar to the a-ketoglutarate dehydrogenase complex (same cofactors, similar substrate and action)

4. Cofactors are the first 4 B vitamins plus lipoic acid: B1 (thiamine; TPP) B2 (FAD) B3 (NAD)

B5 (pantothenate ! CoA) Lipoic acid

Biochem 79 Pentose phosphate pathway

Biochem 80 1. Sites: lactating mammary glands, liver, adrenal cortex

all sites of fatty acid or steroid synthesis 2. Begins with glucose 6-P 3. The irreversible oxidative portion generates NADPH

NADPH needed for: fatty acid and cholesterol (steroid) synthesis, maintaining reduced glutathione inside RBCs

4. The reversible nonxidative portion rearranges the sugars so they can reenter the glycolytic pathway

5. Ribose 5-P, which is needed for nucleotide synthesis, can be formed from glucose 6-P by either arm

6. Major regulatory enzyme: glucose 6-P dehydrogenase Glucose 6-P ! 6-phosphogluconolactone

7. Stimulators: NADP+, insulin 8. Inhibitors: NADPH

Biochem 81 Sucrose and Lactose Metabolism

Biochem 82 1. Sucrase converts sucrose to glucose and fructose

Hexokinase can convert fructose ! fructose 6-P (muscle, kidney)

Fructose enters glycolysis by a different route in the liver Dihydroxyacetone phophate (DHAP) enters glycolysis

directly After glyceraldehyde is reduced to glycerol, it is

phosphorylated and then reoxidized to DHAP 2. Lactase converts lactose to glucose + galactose

Galactokinase converts galactose ! galactose 1-P Galactose 1-P !!! glucose 1-P ! glycolysis

Biochem 83 Glycogen Storage Diseases

Biochem 84 Result in abnl glycogen metabolism & glycogen in cells

DEFFECT TISSUE SIGNS, ETC. Von Gierkes (type I)

Glucose 6-P Liver & kidney

Hepatomegaly, Failure to thrive, Hypoglycemia, Ketosis, Hyperuricemia, Hyperlipidemia

Pompes (type II)

-1,4-glucosidase Lysosomes, All organs

Failure of heart & lungs Death

Biochem 85 Hereditary enzyme deficiencies in lactose and sucrose metabolism

Biochem 86 1. Hereditary enzyme deficiences in sucrose metablism:

Fructokinase deficiency ! essential fructosuria o Benign disorder

Fructose 1-P aldolase deficiency ! hereditary fructose intolerance o Characterized by severe hypoglycemia after ingesting

fructose (or sucrose), jaundice, cirrhosis 2. Inherited enzyme deficiencies in lactose metabolism:

Lactase deficiency ! milk intolerance o Develops in adult life (age-dep) or hereditary (blacks, Asians)

Galactokinase deficiency ! mild galactosemia o Early cataract formation

Galactose 1-P uridyltransferase deficiency ! severe galactosemia (AUTOSOMAL RECESSIVE) o Cataract, hepatosplenomeg, growth failure, retardation, death o Treatment: exclude galactose & lactose from diet

Biochem 87 Pyruvate dehydrogenase deficiency

Biochem 88 1. Pyruvate dehydrogenase deficiency ! neurologic

defects 2. Causes backup of substrate (pyruvate & alanine), resulting

in lactic acidosis 3. Treatment: intake of ketogenic nutrients (Lysine &

Leucine are the only purely ketogenic amino acids)

Biochem 89 Glucose-6-phosphate dehydrogenase deficiency

Biochem 90 1. X-linked recessive 2. Background:

G6PD is the rate limiting enzyme in HMP (hexose monophosphate) shunt, which includes the pentose phosphate pathwy (which yields NADPH)

NADPH is necessary to keep glutathione reduced, which in turn detoxifies free radicals and peroxides

3. Manifestations of disease: NADPH in RBCs ! hemolytic anemia

4. Pathogenesis: Poor RBC defense against oxidizing agents (fava beans,

sulfonamides, primaquine) and antituberculosis drugs 5. More prevalent among blacks 6. Heinz bodies: altered Hemoglobin precipitates w/in RBCs

Biochem 91 Lipid digestion

Biochem 92 1. In mouth:

Medium-chain triacylglycerol (TGs) are hydrolyzed by lipase Continues in stomach, producing a mix of diacylglycerols & FFAs

2. In the duodenum: Lipids are emulsified by bile salts (made from cholesterol in liver)

3. In small intestine: Emulsified fats are hydrolyzed by pancreatic lipase Phospholipids are hydrolyzed by phospholipase A Cholesterol esters are hydrolyzed by cholesterol esterase

4. Mixed micelles form, which contain: Fatty acids Diacylglyc

Monoacyl Phospholipi

Cholesterol VitA,D,E, K

Bile acids

5. Micelles absorbed in small intestine ! further metabolized ! Medium-chain TGs are hydrolyzed Medium-chain fatty acids (8-10 carbons) pass into portal vein Long-chain fatty acids (>12 carbons) are reincorporated into TGs TGs go into chylomicrons ! lymphatics ! circulation via thoracic duct

Biochem 93 How are lipids transported to tissues?

Biochem 94 1. Lipids are transported to tissues in the blood plasma

primarily as lipoproteins: Spherical particles w/a core that contain varying

proportions of hydrophobic triacylglycerols & cholesterol esters

Outer layer of cholesterol, phospholipids, and specific apoproteins

Biochem 95 Lipoprotein absorption

Biochem 96 1. Exogenous lipid (from intestine), except for medium-chain

fatty acids, is released into the plasma as chylomicrons Chylomicrons contain a high proportion of TGs TG is hydrolyzed to FFAs and glycerol by lipoprotein lipase on

the surface of capillary endothelium in muscle and adipose tissue The cholesterol rich chylomicron remnants travel to the liver,

where they are taken up by receptor-mediated endocytosis (RME) 2. Endogenous lipid (from liver) is released into blood as VLDLs

VLDL TG is hydrolyzed by lipoprotein lipase to FFAs and glycerol, yielding low-density lipoproteins (LDLs)

LDLs are removed from circulation by RME in tissues that contain LDL receptors (tissues that need cholesterol, but mostly in liver)

LDL cholesterol: o Inhibits HMG CoA reductase (RLS in cholesterol synthesis) o Down-regulates LDL receptor synthesis ! LDL uptake

High density lipoproteins (HDLs) are made in the liver

Biochem 97 Lipoprotein functions and associated apolipoproteins: Chylomicrons

Biochem 98 1. Delivers dietary triglycerides to peripheral tissues

and dietary cholesterol to liver 2. Secreted by intestinal epithelial cells 3. Excess causes pancreatitis, lipemia retinalis, eruptive

xanthomas 4. Associated apolipoproteins:

B-48 mediates secretion As are used for formation of new HDL C-II activates lipoprotein lipase E mediates remnant uptake by liver

Biochem 99 Lipoprotein functions and associated apolipoproteins: VLDL

Biochem 100 1. Delivers hepatic triglycerides to peripheral tissues 2. Secreted by liver 3. Excess causes pancreatitis 4. Associated apolipoproteins:

B-100 mediates secretion C-II activates lipoprotein lipase E mediates remnant uptake by liver

Biochem 101 Lipoprotein functions and associated apolipoproteins: LDL

Biochem 102 1. Transports hepatic cholesterol to peripheral tissues 2. Formed by lipoprotein lipase modification of VLDL in

peripheral tissue 3. Taken up by target cells via RME 4. Excess causes atherosclerosis, xanthomas, and arcus

corneaa (senilis?) 5. Associated apolipoproteins:

B-100 mediates binding to cell surface receptor for endocytosis

Biochem 103 Lipoprotein functions and associated apolipoproteins: HDL

Biochem 104 1. Mediates centripetal transport of cholesterol (i.e.

reverse cholesterol transport, from periphery to liver, i.e. transports cholesterol from periphery to liver)

2. Acts as a repository for apoC & apoE (which are needed for chylomicron and VLDL metabolism)

3. Secreted from both liver and intestine 4. Associated apolipoproteins:

As help form HDL structure A-I in particular activates LCAT (which catalyzes

esterification of cholesterol) CETP mediates transfer of cholesteryl esters to other

lipoprotein particles

Pneumonic: HDL is Healthy, LDL is Lousy

Biochem 105 Oxidation of fatty acids

Biochem 106 1. Occurs in mitochondrial matrix. The overall process is: RCH2CH2COOH -oxidation CH3COSCoA Citric acid cycle CO2+ H2O 2. Fatty acids must first be activated to their acyl CoA thioesters

Long-chain (LC) fatty acids (>12) activated in cytosol ! LC acyl CoAs are shuttled into mitoch matrix by carnitine transport syst

MCFAs pass directly into the mitoch & are activated in the matrix 3. Fatty acyl CoA is then oxidized to CO2 and H2O by -oxidation:

Continues in cycle until its completely converted to acetyl CoA Each cycle generates 5 ATPs via ETS and 12 ATPs via combined

action of citric acid cycle and ETS Terminal 3 carbons of odd-numbered fatty acids yield propionyl

CoA as the final product, which can: o Enter the citric acid cycle (after carboxylation to succinyl

CoA in a 3-rxn sequence requiring biotin and Vit B12) o Be used for gluconeogenesis

Biochem 107 Ketogenesis

Biochem 108 1. The formation of acetoacetate and -hydroxybutyrate

from metabolism of acetyl CoA in the liver 2. Reaction:

Acetyl CoA + acetoacetyl CoA ! hydroxymethylglutaryl CoA (HMG CoA)

HMG CoA ! acetoacetate and acetyl CoA Acetoacetate ! -hydroxybutyrate (requires NADH)

3. How is acetoacetate used in the body? Extrahepatic tissues (especially heart) can activate

acetoacetate at the expense of succinyl CoA and burn acetoacetyl CoA for energy

Glucose-starved brain can use acetoacetate for fuel (b/c its freely soluble in blood and easily crosses the BBB)

Biochem 109 Fatty acid synthesis

Biochem 110 1. Carried out by fatty acid synthase, a cytosolic complex 2. Primary substrates:

Acetyl CoA: formed in mitoch, mainly by pyruvate dehydrogenase o Its transported to cytosol by citrate-malate-pyruvate shuttle

Malonyl CoA: formed by biotin-linked carboxylation of actyl CoA 3. The acetyl and malonyl moieties are transferred from the sulfur

of CoA to activate sulfhydryl groups in the fatty acid synthase 4. 7 cycles lead to production of palmityl:enzyme, which is

hydrolyzed to yield products palmitate & fatty acid synthase 5. Palmitate is the precursor for longer & unsaturated fatty acids

Chain-lengthening occurs in the mitoch and ER (C16!C18!etc) Desaturating system is also present in the ER

o Requires NADPH and O2 o Inserts double bonds no further than 9 carbons from the

carboxylic acid group

Biochem 111 What do the limitations of the desaturating system result in?

Biochem 112 1. The limitations of the desaturating system impose a

dietary requirement for essential fatty acids (those w/double bonds >10 carbons from the carboxyl end) Lineoleic acid

o Precursor for arachidonic acid (which is beginning of cascade that synthesizes prostaglandins, thromboxanes, and eicosanoids)

Linolenic acids

Biochem 113 Glycerolipid synthesis

Biochem 114 1. This process is carried out by the liver, adipose tissue,

and the intestine 2. Pathways begin w/glycerol 3-P, which is mainly produced by

reducing dihydroxyacetone phosphate w/NADPH 3. Succesive transfers of acyl groups from acyl CoA to carbons 1

and 2 of glycerol 3-phosphate produce phosphatidate, which can then be converted to a variety of lipids: Triacylglycerol (from transfer of acyl group from acyl CoA) Phosphatidyl choline & phosphatidyl ethanolamine (from

transfer of base from its cytidine diP/CDP derivative) Phosphatidylserine (from exchange of serine for choline) Phosphatidylinositol (from reaction of CDP-diacylglycerol

with inositol)

Biochem 115 Sphingolipid synthesis

Biochem 116 1. Begins with palmityl CoA and serine

Produces dihydrosphingosine and sphingosine 2. Sphingosine can then by acylated to produce ceramide

Additional groups may be added to the C1-OH of ceramides

Biochem 117 Cholesterol synthesis

Biochem 118 1. Cholesterol is made by the liver and intestinal

mucosa from acetyl CoA in a multistep process 2. Key intermediate = HMG CoA

HMG CoA reductase: regulatory enzyme that catalyzes HMG CoA + NADPH ! mevalonic acid

Increasing amounts of intracellular cholesterol lead to inhibition of HMG CoA reducate and accelerated degradation of the enzyme

3. Overall reaction: Acetoacetyl CoA + acetyl CoA HMG CoA synthase ! HMG CoA

HMG CoA reductase ! mevalonic acid !!!cholesterol

Biochem 119 What are the fates of the products of cholesterol synthesis?

Biochem 120 1. Mevalonic acid

Precursor of a number of natural products called terpenes, which include vit A, vit K, coenzyme Q, and natural rubber

2. Cholesterol Converted to steroid hormones in the adrenal cortex,

ovary, placenta, and testes Majority is oxidized to bile acids in the liver 7-dehydrocholesterol is the starting point for synthesis

of vit D

Biochem 121 Lipid malabsorption

Biochem 122 1. Leads to excessive fat in the feces (steatorrhea) 2. Occurs for a variety of reasons:

Bile duct obstruction o ~50% of dietary fat appears in the stools as soaps

(metal salts of LCFAs) o Absence of bile pigments leads to clay-colored stools o Deficiency of the ADEK vitamins may result

Pancreatic duct obstruction o Stool contains undigested fat o Absorption of ADEK vitamins is not sufficiently

impaired to lead to deficiency symptoms Diseases of the small intestine (e.g., celiac disease,

abetalipoproteinemia, nontropical sprue, IBD)

Biochem 123 Hyperlipidemias

Biochem 124 1. Familial hypercholesterolemia

Results from defective LDL receptors Findings: severe atherosclerosis, early death from CAD Tx: HMG CoA reductase inhibitors (statins)

2. Hypertriglyceridemia Can result from either overproduction of VLDL or defective

lipolysis of VLDL triglycerides Findings: cholesterol levels may be mildly

3. Mixed hyperlipidemias BOTH serum cholesterol & serum triglycerides are There is both overproduction of VLDL and defective

lipolysis of triglyceride-rich lipoproteins (VLDL and chylmicrons)

There is a danger of acute pancreatitis

Biochem 125 Inheritied defects and deficiencies that disrupt fatty acid oxidation

Biochem 126 1. Inherited defects in the carnitine transport system,

which have widely varying symptoms: Hypoglycemia Muscle wasting w/accumulation of fat in muscle Feeding fat w/medium-chain triacylglycerols (e.g., butterfat)

is helpful in some cases, b/c MCFAs can bypass carnitine transport system

2. Inherited deficiencies in the acyl CoA dehydrogenase, the most common being medium-chain (C6-C12) acyl CoA dehydrogenase deficiency Hypoketotic hypoglycemia and dicarboxylic aciduria occur,

with vomiting, lethargy, and coma This is believed to account for the condition called Reye-

like syndrome

Biochem 127 Sphingolipid Storage Diseases

Biochem 128

Disease Accumulated

Substance Clinical Manifestations

Tay-Sachs Ganglioside GM2 Mental retardation (MR), blindness, red spot on macula, death by 3rd yr Gauchers Glucocerebroside Hepatosplenomeg, bone erosion, MR

Fabrys Ceramide trihexoside Rash, kidney failure, lower extremity pain Niemann-Pick Sphingomyelin Hepatosplenomegaly, MR Globoid cell

leukodystrophy Galactocerebroside MR, myelin absent

(also called Krabbes disease) Metachromatic leukodystrophy

Sulfatide MR, metachromasia, nerves stain yellowish brown w/crystal violet

Gen gangliosidosis Ganglioside GM1 MR, hepatomegaly, skeletal abnormalities

Sandhoffs Ganglioside GM2, globoside Same as Tay-Sachs, but more rapid course

Fucosidosis Pentahexosylfuco-glycolipd Cerebral degeneration, spasticity, thick skin

Biochem 129 Urea Cycle

Biochem 130 1. Converts NH4+ (which is toxic, esp to CNS) to urea 2. Occurs in the liver 3. Urea is excreted in the urine 4. NH4+ + CO2 carbamoyl phosphate synthetase I !

carbamoyl P + ornithine ornithine transcarbamoylase ! citrulline + aspartate + ATP argininosuccinate synthetase ! argininosuccinate argininosuccinate lyase ! fumarate + arginine + H20 arginase ! UREA + ornithine

Urine byproduct

Biochem 131 How does detoxification of NH4+ occur in peripheral tissues?

Biochem 132 1. In most tissues:

Glutamine synthetase incorporates NH4+ into glutamate to form glutamine, which is carried by circulation to the liver

In the liver, glutaminase hydrolyzes glutamine back to NH4+ and glutamate

2. In skeletal muscle: Glutamate dehydrogenase and glutamate-pyruvate

aminotransferase ! incorporate NH4+ into alanine Alanine is carried to the liver, where transdeamination

results in converstion of alanine back to pyruvate and NH4+

Biochem 133 Hyperammonemia

Biochem 134 1. May be caused by insufficent removal of NH4+, resulting

from disorders that involve one of the enzymes in the urea cycle 2. Signs and Symptoms

Blood NH4+ concentrations above the normal range (30-60 M) Mental retardation, seizure, coma, and death

3. Enzyme defects Low activity of carbamoyl P synthetase or ornithine-carbamoyl

transferase ! [NH4+] in blood & urine ! NH4+ intoxication Defective argininosuccinate synthetase, argininosuccinase, OR

arginase ! blood levels of metabolite preceding defect o NH4+ levels may also rise

4. Treatment Restriction of dietary protein Intake of mixes of keto acids that correspond to essential amin acid Eating benzoate & phenylacetate: alternate path for NH4+ excretion

Biochem 135 Carbon skeletons of amino acids

Biochem 136 1. Amino acids can be grouped into families based on the point

where their carbon skeletons enter the TCA cycle 2. AcetylCoA/Ketogenic fam(blue:keto-& glucogenic; red:ketogen only)

Isoleucine, leucine, lysine, phenylalanine, tryptophan, and tyrosine Phenylalanine ! tyrosine via phenylalanine hydroxylase Tyrosine is starting compound for:

o Epi and NE, T3 and T4, Dopamine, Melanin 3. -Ketoglutarat fam (arginine,histidine,glutamate,gluatmine,proline)

Histidine precursor of histamine Glutamate excitatory neurotransm, can be converted to GABA

4. Succinyl CoA family (isoleucine, methionine, valine) Methyl of methionine participates in methylation rxns as S-

adenosylmethionine (SAM) 5. Fumarate family (phenylalanine and tyrosine) 6. Oxaloacetate family (asparagine and aspartate) 7. Pyruvate fam (alanine,cysteine,glycine,serine,threonine, tryptophan)

Biochem 137 Essential amino acids

Biochem 138 1. Isoleucine 2. Leucine 3. Lysine 4. Phenylalanine 5. Tryptophan 6. Histidine 7. Methionine 8. Valine 9. Threonine

Biochem 139 Phenylketonuria (PKU)

Biochem 140 1. Results from a deficiency of:

Phenylalanine (Phe) hydroxylase OR Dihydropteridine reductase

2. Findings Phe in the blood (hyperphenylalaninemia) Phe builds up to toxic concentrations in body fluids,

resulting in CNS damage with mental retardation Phe inhibits melanin synthesis ! hypopigmentation

3. An alternative pathway for Phe breakdown produces phenylketones, which spill into th eurine

4. In those affected, tyrosine is an essential amino acid 5. Treatment: restricting dietary protein (phenylalanine)

Biochem 141 Albinism

Biochem 142 1. No tyrosinase (1st enzyme on pathway to melanin) 2. Have little or no melanin and are:

Easily sunburned Very susceptible to skin carcinoma Photophobic b/c of lack of pigment in iris of eye

Biochem 143 Homocystinuria

Biochem 144 1. May result from several defects:

Cystathionine synthase (CS) deficiency affinity of CS for its coenzyme, pyridoxal phosphate (PLP)

(may respond to megadoses of pyridoxine/vit B6) Methyl tetrahydrofolate homocyst methyltransferase deficiency Vit B12 coenzyme deficiency (may respond to vit B12)

2. Finding: homocysteine accumulation in blood, appears in urine 3. Pathologic changes

Dislocation of optic lens Mental retardation Osteoporosis and other skeletal abnormalities Atherosclerosis and thromboembolism

4. Pts unresponsive to vitamin therapy may be treated with: Synthetic diets low in methionine Betaine (trimethylglycine) alternative methyl group donor

Biochem 145 Maple-syrup urine disease

Biochem 146 1. Results from a deficiency in the branched-chain 2-

keto acid decarboxylase 2. Findings: branched chain keto acids derived from

isoleucine, leucine, and valine appear in the urine, giving it a maple syrup-like odor

3. Elevated keto acids cause severe brain damage, with death in the first year of life

4. Treatment A few respond to megadoses of thiamine (vitamin B1) Those that dont: synthetic diets low in branched-chain

amino acids

Biochem 147 Histidinemia

Biochem 148 1. Deficiency in histidine--deaminase (the 1st enzyme

in histidine catabolism) 2. Characterized by elevated histidine in blood plasma and

excessive histidine metabolites in urine 3. Symptoms:

Mental retardation and speech defects (both are rare) 4. Treatment: not usually indicated

Biochem 149 Origin of the atoms in the purine ring

Biochem 150

N3, N9: glutamine

N

N-formyl tetrahydrofolate

N

C

C C

C

NH

C

N

C4, C5, N7: glycine

N-formyl tetrahydrofolate

aspartate

C6: respiratory CO2

N3, N9: glutamine

Biochem 151 PURINE nucoleotide synthesis

Biochem 152 De novo synthesis:

1. Inosine monophosphate (IMP), AMP, & GMP inhibit PRPP synthetase 2. Committed step: conversion of PRPP to 5-phosphoribosyl-1-amine

PRPP activates glutamine PRPP amidotransferase Inhibited by end products (IMP, GMP, AMP) of the pathway

Purines made by salvage of preformed purine bases: 1. Involves 2 enzymes:

Hypoxanthine-guanine phophoribosyltransferase (HGPRT) o Comp inhibited by IMP and GMP

Adenine phosphoribosyl transferase o Inhibited by AMP

PATHWAY: (remember: purines = adenine and guanine) Ribose 5-P PRPP synthetase! PRPPglutamine PRPP amidotransferase! 5-phosphoribosyl-1-amine 9 rxns! IMP + Asp + GTP ! AMP

! IMP + Gln + ATP + NAD ! GMP

Biochem 153 Regulation of purine synthesis

Biochem 154 1. Regulation provides a steady supply of purine

nucleotides 2. GMP and AMP inhibit 1st step in their own synthesis from IMP 3. Reciprocal substrate effect: GTP is a substrate in AMP

synthesis, and ATP is a substrate in GMP synthesis Balances supply of adenine and guanine ribonucleotides

4. Interconversion among purines ensures control of their levels AMP deaminase converts AMP back to IMP GMP reductase converts GMP back to IMP IMP is the starting point for synthesis of AMP and GMP

Biochem 155 Origin of the atoms in the pyrimidine ring

Biochem 156

N

N

C

C C

C

C2, N3: carbamoyl phosphate C4, C5, C6, N1: aspartate

Biochem 157 De novo pyrimidine synthesis

Biochem 158 1. In mammals, 1st 3 steps occur on one multifunctional

enzyme called CAD, which stands for the names of the enzymes CO2 + glutamine CAP synthetase II ! carbamoyl-P (CAP) Synthesis of dihydroorotic acid is a 2-step process:

o Committed step: aspartate + CAP aspartate transcarbamoylase ! carbamoyl aspartate

o Carbam aspartate dihydrorotase! dihydroorotic acid + H2O 2. Dihydroorate forms UMP

Dihydroorate "! orotic acid Orotic acid + PRPP ! orotidylate (OMP) Decarboxylation of OMP ! uridylate (UMP) These 2 steps occur on 1 protein (if defected: orotic aciduria)

3. Synthesis of remaining pyrimidines involves UMP Phosphorylation of UMP ! UDP + UTP Addition of amino group from glutamine to UTP ! CTP

Biochem 159 Regulation of pyrimidine synthesis

Biochem 160 1. CAP synthetase II regulation

Inhibited by UTP Activated by ATP and PRPP

2. CTP itself inhibits CTP synthetase

Biochem 161 Salvage of pyrimidines

Biochem 162 Accomplished by the enzyme pyrimidine phosphoribosyl transferase, which can use orotic acid, uracil or thymine, but NOT CYTOSINE

Biochem 163 Deoxyribonucleotide synthesis

Biochem 164 Formation of deoxyribonucleotides (for DNA synthesis) involves reduction of sugar of ribonucleoside diphosphates: 1. Ribonucleotide reductase

Leads to reduction of ADP, GDP, CDP, or UDP to deoxyribonucs Its reducing power is from 2 sulfhydryl groups on thioredoxin Using NADPH + H+, thioredoxin reductase converts oxidized

thioredoxin back to the reduced form Regulation controls the overall supply of deoxyribonucleotides

o Rxn proceeds only in presence of nucleotide triphosphate o dATP: allosteric inhibitor o Other deoxynucleosides interact w/enzyme to alter specificity

2. Thymidylate synthase Catalyzes formation of dTMP (deoxythymidylate) from dUMP Coenzyme: N5, N10-methylene tetrahydrafolate (regenerated after

each rxn by dihydrofolate reductase)

Biochem 165 Nucleotide degradation

Biochem 166 1. Purine degradation (product: Uric acid is exreted in urine)

Sequential actions of 2 groups of enzymes (nucleases and nucleotidases) lead to hydrolysis of nucleic acids to nucleosides

Deaminase converts adenosine/deoxyadenosine to deoxy-/inosine Purine nucleoside phosphorylase splits inosine and guanosine to

ribose 1-P and free bases hypoxanthine and guanine Guanine is deaminated to xanthine Hypoxanthine & xanthine xanthine oxidase! uric acid

2. Pyrimidine degradation (products = -amino acids, CO2, NH4+) Degraded to free bases uracil or thymine A 3-enzyme rxn (reduction, ring opening, deamination-

decarboxylation) converts uracil to CO2, NH4+, and -alanine and thymine to CO2, NH4+, & -aminoisobutyrate

THUS: urinary -aminoisobutyrate is an indicator of DNA turnover (may be during chemo or radiation therapy)

Biochem 167 Disorders caused by deficiencies in enzymes involved in nucleotide metabolism

Biochem 168 Hereditary orotic aciduria

Orotate phosphoribosyl transferase and/or OMP decarboxylase

Retarded growth, Anemia

Synthetic cytdine or uridine (UTP formed acts as inhib of carbamoy-P synthetase II)

Purine phosphorylas deficiency

purine uric acid formation

Impaired T-cell function

SCID Adenosine deaminase

T- & B-cell dysfunction w/early death from infection

Gene therapy

Lesch-Nyhan HGPRT (deficient or absent)

purine synthesis, hyperuricemia, severe neuro problem (spastic, MR, self-mutilation)

Allopurinol - deposition of sodium urate crystals, but doesnt ameliorate neuro symptoms

Biochem 169 Anticancer drugs that interfere w/nucleotide metabolism

Biochem 170 1. Hydroxyurea

Inhibits nucleoside diphosphate reductase (enzyme that converts ribonucleotides to deoxyribonucleotides)

2. Aminopterin and methotrexate Inhibit dihydrofolate reductase (enzyme that converts

dihydrofolate to tetrahydrofolate) 3. Fluoredeoxyuridylate

Inhibits thymidylate synthetase (enzyme that converts dUMP to dTMP)

Biochem 171 Gout

Biochem 172 1. May result from a disorder in purine metabolism 2. Is associated w/hyperuricemia 3. Primary gout: overproduction of purine nucleotides

Mutations in PRPP synthetase ! loss of feedback inhibition by purine nucleotides

A partial HGPRT deficiency ! less PRPP is consumed by salvage enzyes ! PRPP activates PRPP amidotransferase

4. Secondary gout Due to radation therapy, CA chemo (b/c they cell death)

5. Treatment: allopurinol Xanthine oxidase catalyzes oxidation of allopurinol to

alloxanthine, which is a potent inhibitor of the enzyme Result: uric acid levels fall, hypoxanthine & xanthine levels

rise (is OK, b/c they dont form crystals)

Biochem 173 Energy expenditure (3 components)

Biochem 174 1. Basal energy expenditure (BEE)

resting energy expenditure Energy used for metabolic processes at rest Represents >60% of total energy expenditure Related to the lean body mass

2. Thermic effect of food Energy required for digesting and absorbing food Amounts to ~10% of energy expenditure

3. Activity-related expenditure 20-30% of daily energy expenditure

Biochem 175 Caloric yield from foods and what % they should be in diet

Biochem 176 1. Carbs: 4 kcal/g

50-60% of caloric intake 2. Proteins: 4 kcal/g

10-20% of caloric intake (0.8 g/kg body weight/day) 3. Fats: 9 kcal/g

No more than 30% of caloric intake 4. Alcohol: 7 kcal/g

Biochem 177 Fats: Essential fatty acids, Deficiency, and Excess storage

Biochem 178 1. Essential fatty acids (EFAs):

Linoleic acid Linolenic acid

2. Deficiency Mainly seen in low-birth-weight infants maintained on

artificial formulas and adults on TPN Characteristic system: scaly dermatitis

3. Excess fat Stored as triacylglycerol

Biochem 179 Marasmus vs. Kwashiorker

Biochem 180 Marasmus Kwashiorker

Insufficient food, including both calories and protein

Starvation with edema often due to protein deficient diet

Depleted subQ fat Pitting edema Flaky paint dermatosis: dark patches on skin that peel

Liver ketogenesis!brain&heart fuel Large liver due to fatty infilatration Muscle wasting (break protein for gluconeogenesis & protein synthes)

Muscle wasting less severe

Frequent infections Frequent infections Low body temp Micronutrient deficiencies Other nutrient deficiencies Slowed growth(60% expected wgt) Death when energy & protein reserves exhausted

Poor appetitie (anorexia) Watery stools w/undigested food Mental changes (apathetic)

Biochem 181 Vitamin A

Biochem 182 1. Functions:

11-cis-retinal prosthetic group of rhodopsin Beta-carotene antioxidant NOT TOXIC at high doses Retinyl phosphate mannose acceptor/donor in glycoprotein synth Retinol & retinoic acid regulate tissue growth & differentiation

2. Deficiency signs and symptoms: Night blindness, Xerophthalmia (cornea keratinizes: Bitot spots)

o Leading cause of child blindness in 3rd world nations Follicular hyperkeratosis (rough, tough skin) Anemia resistance to infection susceptibility to cancer

3. Toxicity (prolonged ingestion of 15,000-50,000 equivalents/day) Bone pain, scaly dermatitis, hepatosplenomegaly, nausea, diarrhea

4. Clinical usage: For acne and psoriasis

Biochem 183 Vitamin D

Biochem 184 1. Functions: regulation of Ca+ metabolism

Stimulates synth of Ca+-binding protein ! aids absorption In combo w/PTH, blood Ca+ by:

o bone demineralization by stimulating osteoblastic activity o Simulates Ca+ reabsorption by distal renal tubules

2. Sources: Major: skin (UV: 7-dehydrocholesterol ! Vit D3/cholecalciferol) Diet (vit D3) and foods fortified w/vit D2

3. Activation Liver: Vit D3 ! 25(OH)D3 Kidney: 25(OH)D3 ! active 1,25(OH)2D3 (stimulated by PTH)

4. Deficiency Rickets (kids): soft bones, stunted growth Osteomalacia (adults): pathologic fractures Bone demineraliz may also result from vit D ! inactive forms by steroids

5. Toxicity: (hyperCa+, metast calcification, bone demineraliz, kidney stones) Seen in sarcoidosis (epithelioid macroph convert vit.D to its active form)

Biochem 185 Vitamin E

Biochem 186 1. Function

Protection of membranes & proteins from free-radical damage Includes isomers of tocopherol:

o Tocopherol + free radicals ! tocopheroxyl radical ! vit C reduces tocopheroxyl radical ! tocopherol is regenerated

2. Deficiency Secondary to impaired lipid absorption (cystic fibrosis, celiac

disease, chronic cholestasis, pancreatic insufficiency, abetalipoproteinemia)

Signs & Symptoms: o Ataxia o Impaired reflexes o Myopathy o Muscle weakness o Hemolytic anemia (b/c of fragility of RBCs) o Retinal degeneration

Biochem 187 Vitamin K

Biochem 188 1. Function

Post-translational carboxylation of glutamyl residues in Ca+-binding proteins: factors VII, IX, & X

2. Deficiency ( PT, aPTT, but nl bleeding time) Impaired blood clotting ! bruising, bleeding Causes:

o Fat malabsorption o Drugs that interfere w/vit K metabolism (warfarin) o Antibiotics that suppress bowel flora

3. Vitamin K in infants Neonates are born w/low stores of vit K Vit K crosses placental barrier poorly Newborns given single injection of vit K High doses: anemia, hyperbilirubinemia, kernicterus

Biochem 189 The B vitamins

Biochem 190 1. B1 = Thiamine 2. B2 = Riboflavin 3. B3 = Niacin 4. B5 = Pantothenate (pantothenic acid) 5. B6 = Pyridoxine (pyridoxamine, pyridoxal) 6. B12 = cobalamin

Biochem 191 Thiamine (vitamin B1)

Biochem 192 1. Thiamine pyrophosphate (TPP): required for nerve

transmission & is coenzyme for several key enzymes: Pyruvate & -ketoglutarate dehydrogenase(glycolysis, TCA) Transketolase (pentose phosphate pathway) Branched-chain keto-acid dehydrogenase (valine, leucine,

isoleucine metabolism) 2. Deficiency leads to beriberi, which occurs in 3 stages:

Early: loss of appetite, constipation, nausea, periph neuropathy, irritability, fatigue

Moderately severe: Wernicke-Korsakoff syndrome (mental confusion, ataxia, ophthalmoplegia)

Severe (in addtion to polyneuritis): o Dry: atrophy & weakness of muscles o Wet:edema,high-output cardiac failure,pulm congestion

Biochem 193 Riboflavin (vitamin B2)

Biochem 194 1. Function:

Converted to re-dox coenzymes FAD & FMN 2. Deficiency signs & symptoms:

Angular cheilitis Glossitis (red and swollen tongue) Scaly dermatitis (esp at nasolabial folds & around

scrotum) Corneal vascularization

Biochem 195 Niacin (vitamin B3)

Biochem 196 1. Function:converted to redox coenzymes NAD & NADP 2. Deficiency

Causes: o Hartnup disease o Malignant carcinoid syndrome o INH

Mild deficiency: glossitis Severe deficiency: pellagra the 3 Ds

o Dermatitis o Diarrhea o Dementia

3. High doses Vasodilation (very rapid flushing) Metobolic changes: blood cholesterol & LDLs

Biochem 197 Vitamin B6 (pyridoxine, pyridoxamine, & pyridoxal)

Biochem 198 1. Function

Coenzyme involved in transamination (e.g., ALT & AST), decarboxylation, and trans-sulfuration (rxns of amino acid metabolism)

2. Deficiency (inducible by INH) Mild: irritability, nervousness, depression Severe: periph neuropathy, convulsions, occasional

sideroblastic anemia Other symptoms: eczema, seborrheic dermatitis around ears,

nose, and mouth; chapped lips; glossitis; angular stomatitis 3. Clinical usefulness:

High doses: tx homocystinuria (defective cystathione -synthase)

Biochem 199 Vitamin B6: Pantothenic acid

Biochem 200 1. Function

Essential component of coenzyme A (CoA) and of fatty acid synthase

Cofactor for acyl transfers 2. Deficiency (very rare)

Vague presentation, little concern to humans Dermatitis, enteritis, alopecia, adrenal insufficiency

Biochem 201 Biotin

Biochem 202 1. Function

Covalently linked biotin = prosthetic group for carboxylation enzymes (e.g. pyruvate carboxylase, acetyl CoA carboxylase) (NOT decarboxylations)

2. Deficiency (rare) Signs and symptoms:

o Dermatitis o Hair loss o Atrophy tongue papilla o Gray mucous memb

o Paresthesa,muscle pain o Hypercholesterlemia o ECG abnormalities

Causes o Antibiotic use (since intestinal bacteria make biotin) o Eating Avidin (raw egg whites)

Binds biotin in a nondigestible form If you eat >20 eggs/day

Biochem 203 Folic acid

Biochem 204 1. Function

Polyglutamate derivatives of tetrahydrofolate serve as coenzymes in 1-carbon transfer rxns: o Purine & pyrimidine synthesis o Thymidylate synthesis o Conversion of homocysteine to methionine o Serine-glycine interconversion

2. Deficiency Signs & symptoms:

o Megaloblastic anemia o Neural tube defects o blood homocysteine associated w/atherosclerotic disease

Can be caused by several drugs: o Methotrexate (chemo) o Trimethoprim (antibact) o Pyrimethamin(antimalari)

o Diphenylhydantoin (anticonvulsant)

o Primidone (anticonvuls)

Biochem 205 Vitamin B12 (cobalamin)

Biochem 206 1. Functions

Coenzyme for methylmalonyl CoA ! succinyl CoA (methylmalonyl CoA mutase) in propionyl CoA metabolism

Coenzyme for methyl transfer between tetrahydrofolate & methionine (homocysteine methyl transferase)

2. Deficiency: Signs & Symptoms:

o Megloblastic anemia o Paresthesia, optic neuropathy, subacute combined degenerat o Prolonged deficiency ! irreversible nervous system damage

Causes: o Intake of no animal products (vegans) o Achlorhydria, intrinsic factor (impaired absorption) o Malabsorption (impaired pancreatic function, sprue, enteritis,

D. latum, absence of terminal ileum/Crohns) 3. Use Schilling test to detect deficiency

Biochem 207 Vitamin C (ascorbic acid)

Biochem 208 1. Functions

Coenzyme for re-dox rxns, including: o Post-translational hydroxylation of proline & lysine in

maturation of collagen o Carnitine synthesis o Tyrosine metabolism o Catecholamine neurotransmitter synthesis

Antioxidant Facilitator of iron absorption

2. Deficiency Signs & symptoms:

o Mild: capillary fragility w/easy bruising & petechiae (pinpoint hemorrhages in skin), immune function

o Severe: scurvy ( wound healing, osteoporosis, hemorrhage, anemia, swollen gums, teeth may fall out)

Biochem 209 y Symptoms of Mineral Deficiencies

Biochem 210 Mineral Deficiency-Associated Conditions

Calcium Paresthesia Tetany Bone fractures, bone pain Osteomalacia (as in vit D deficiency)

Iodine Goiter Cretinism

Iron Anemia Fatigue, tachycardia, dyspnea

Magnesium Neuromusc excitability, paresthesia Depressed PTH release

Phosphorus (as phosphate) Deficiency rarely occurs Zinc Growth retardation & hypogonadism

Dry, scaly skin Mental lethargy Imparied taste & smell, poor appetite

Biochem 211