Biochem Review[1]

Biochemistry Review for the NBDE 2008 November 5, 2008

Janeen Arbuckle, PhD

Contact Information:

[email protected]

Course Objective Review the biochemical processes most frequently tested on the NBDE

General Outline- 1st hour

Brief overview of Enzyme KineticsEnzyme Regulation and Inhibition Carbohydrate Structure and Function

enz (z) A B

Michaelis-Menton Kinetics [substrate] vs. Vo is hyperbolic At high [substrate] the reaction reaches its maximum velocity (Vmax) The Km is the [substrate] at which the reaction velocity is Vmax The Km is an effective measure of affinity.

The Km is a Measure of Affinity

Km is inversely related to affinity

Enzymes with a low Km have a high affinity for substrate Enzymes with a high Km have a low affinity for substrate

Hexokinase vs GlucokinaseHexokinase- Ubiquitously expressed High affinity for glucose Low Km and Vmax Saturated at low [glucose] Glucokinase- Liver specific enzyme High Km and Vmax Saturated at high [glucose]

Hexokinase functions during fasting Glucokinase functions after a glucose rich meal

Regulation of Enzyme Activity Allosteric EffectorsThe activity of allosteric enzymes can be regulated by molecules that bind to sites on the enzyme other than the active site. Allosteric enzymes commonly contain multiple subunits and catalyze rate-limiting steps. Phosphofructokinase-1 (+) allosteric regulators: AMP, and Fructose, 2-6 bisphosphate (-) allosteric regulators: ATP and Citrate

Regulation of Enzyme Activity Covalent ModificationOne of the primary means of modifying enzyme activity is by the addition or removal of a phosphate group. Phosphates are typically added by kinases and removed by phosphatases. Enzymes can either be activated or inactivated by the addition phosphate group. glycogen phosphorylase is active when phosphorylated glycogen synthase is inactive when phosphorylated

of a

Enzyme InhibitionCompetitive vs. Noncompetitive Competitive Inhibitors bind the same site on the enzyme as the substrate increase the Km of the enzyme do not affect the Vmax

Increasing the substrate concentration will eventually out-compete the inhibitor.

Competitive InhibitorsVmax is unchanged in the presence of a competitive inhibitor

Km is increased in the presence of a competitive inhibitor

Km is increased

Vmax is unchanged

Enzyme InhibitionCompetitive vs. Noncompetitive Noncompetitive Inhibitors do not bind the same site as the substrate do not affect the Km of the enzyme decreases the Vmax

The reaction will never reach the original maximum velocity, regardless of the substrate concentration

Noncompetitive InhibitorsVmax is decreased in the presence of noncompetitive inhibitor

Km is unchanged in the presence of a noncompetitive inhibitor

Km is unchanged

Vmax is decreased

Enzyme Inhibition and Regulation Takeaway PointsCompetitive Inhibitors Increase the Km without affecting the Vmax Noncompetitive Inhibitors Do not affect the Km but decrease the Vmax Enzymes are regulated by allosteric effectors e.g. citrate is a negative regulator of PFK-1 covalent modification e.g. phosphorylation of glycogen phosphorylase activates the enzyme

Part II figure-3

CarbohydratesGeneral formula (CH2O)n Functions: Immediate Energy Source Blood Glucose Energy Stores Extracellular Matrix Glycoconjugates Nucleic Acid Precursor Glycogen (homopolymer) Glycosaminoglycans (heteropolymer) Glycoproteins Glycolipids Ribose

CarbohydratesDisaccharides Lactose = Sucrose = Maltose = Polysaccharides Starchamylose (14 linkage) amylopectin, branched (14, 16) polysaccharides of glucose (16, 13, and 14) glucose + galactose glucose + fructose glucose + glucose

DextranLehninger Figure 1-10dStructural Isomers Glucose Galactose Fructose

Product of oral bacteria Found in dental plaqueGlycosaminoglycans- Hyaluronic Acid Chondroitin Sulfate

Monosaccharides Can be in an open chain form or a cyclic form In the open chain form, each has a carbonyl.

Six carbon sugars tend to be cyclic Cyclization generates an anomeric carbon isomers, and Lehninger Figure 7-6

two

Sugars with free anomeric carbons are reducing sugars

Disaccharides

Lactose = galactose(14) glucose Anomeric carbon for galactose is C-1 Anomeric carbon for glucose is C-1 C-1 of glucose remains free Lactose is a reducing sugar Reducing End of Lactose

Sucrose = fructose

glucose(12)

Anomeric carbon for glucose is C-1 Anomeric carbon for fructose is C-2 No free anomeric carbon Sucrose is not a reducing sugar Lehninger Figure 7-12

Starch

Storage form of glucose in plants Is made of amylose and amylopectin Amylose polymerized glucose no branches (14) linkage

(14)

Lehninger Figure 7-15

Amylopectin polymerized glucose branched (14) linkage (16) at branch points many reducing ends for quick mobilization

GlycogenPrimary linkage (14)

Branch point linkage (16)

Storage form of glucose in humans Primarily stored in liver functions to maintain blood glucose levels Stored in muscle functions to provide glucose during exercise


Heteropolysaccharides GlycosaminoglycansGAGs are long, unbranched heteropolysaccharide chains composed of repeating disaccharide units. Modified by negatively charged carboxyl and sulfate groups Extended, unbranched chains surrounded by a shell of water gel-like matrix or ground substance Neighboring molecules tend to repel each other slippery nature of mucous secretions and synovial fluid Compressible but rebound after the release of pressure resilience of synovial fluid and vitreous humor GAGs vary based on their disaccharide subunits, linkage, sulfation patterns and distribution in the human body. Majority are linked to a core protein to form a proteoglycan

Chondroitin SulfateMost abundant GAG in the body Found in cartilage, tendons, ligaments and aorta Forms proteoglycan aggregates with hyaluronic acid.

Hyaluronate/Hyaluronic AcidDifferent from other GAGs unsulfated not covalently attached to proteins Is a lubricant and shock absorber Lehninger Figure 7-24 Found in synovial fluid, vitreous humor, loose connective tissue

Additional GAGsKeratin Sulfate present in cornea and connective tissue Dermatan Sulfate found in skin, blood vessels and heart valves Heparin

the only intracellular GAGfound in mast cells lining arteries of liver, lungs and skin functions as an anticoagulant Heparin Sulfate extracellular found in basement membranes

Proteoglycans

All GAGs, except for hyaluronate, are covalently attached to proteins to form proteoglycans. In cartilage, a core protein is linked to linear GAG chains. The negative charge of GAGs causes repulsion between molecules, generating a bottle brush appearance

ProteoglycansSugar Content >> Protein Content

MucopolysaccharidosesGAGs are normally degraded in lysosomes by specific enzymes Deficiency in any of these enzymes leads to accumulation of GAGs and a variety of symptoms such as skeletal and extracellular matrix deformities and mental retardation Hurler Syndrome -L-Iduronidase deficiency sx- corneal clouding mental retardation dwarfing Iduronate sulfatase deficiency X-linked inheritance sx- NO corneal clouding physical deformity mental retardation

Hunter Syndrome

GlycoproteinsProteins modified by oligosaccharides Differ from proteoglycans carbohydrate chain is shorter, branched, fewer negative charges Function in cell surface recognition cell surface antigenicity (e.g. blood group antigens) extracellular matrix mucins of GI/GU tracts (biological lubricants)

Glycoproteins (contd)O-glycosidic bonds carbohydrate is attached to hydroxyl group of serine or threonine N-glycosidic bonds carbohydrate is attached to the amide group of an asparagine side chain Occur as post-translational modifications the

Carbohydrates Takeaway PointsMonosaccharides Disaccharides Polysaccharides Homopolysaccharides Heteropolysaccharides Starch Glycogen GAGs

Extended linear repetitions of disaccharides Central components of the extracellular matrix Hyaluronic Acid, Chondroitin Sulfate Glycoproteins Post-translational glycosylation of serine, threonine or asparagine residues Function in cellular recognition, cell surface antigenicity

Course Objective Review the biochemical processes most frequently tested on the NBDE

General Outline- 2nd hour Carbohydrate Metabolism Glycolysis Krebs Cycle Quiz

Digestion of Carbohydrates Begins in the mouth - amylase breaks (14) bonds of starch and glycogen oligosaccharides In the small intestine - amylase produced by the pancreas further digests starch and glycogen oligo- and di-saccharidases made by intestinal mucosal cells catalyze the final digestion Small intestine absorbs

monosaccharides

Major Pathways of Glucose Utilization

GlycolysisAnaerobic Cytosolic

Two phases Preparatory (requires energy ) Pay-off (produces energy)

Kinase: Isomerase: Aldolase:

Phosphorylates Rearranges Cleaves C-C figure-14-2a

- 2 ATP per glucose

GlycolysisAnaerobic CytosolicTwo phases Preparatory - 2 ATP per glucose Pay-off + 4 ATP per glucose Net 2 ATP per glucose

+ 4 ATP per glucose

figure-14-2b

Glucose Glucose-6-PhosphateATP ADP

Regulation of GlycolysisPhosphorylation of Glucose Phosphorylation of Fructose-6-Phosphate Generation of PyruvatePhosphorylation of Glucose commits glucose to inside cell two isoforms (Hexokinase vs Glucokinase) hormonal regulation

Hexokinase vs GlucokinaseHexokinase- Ubiquitously expressed High affinity for glucose Low Km and Vmax Saturated at low [glucose] Glucokinase- Liver specific enzyme High Km and Vmax Saturated at high [glucose]

Liver has a selective advantage for the uptake of glucose

Regulation of GlycolysisPhosphorylation of Glucose Phosphorylation of Fructose-6-Phosphate Generation of Pyruvate

Phosphofructokinase-1 Rate-Limiting Step Commits Glucose to Glycolysis Allosterically Regulated (-) ATP, citrate (+) AMP, F2,6BP

Fructose-6-PhosphateATP

ADP

Fructose-1,6-Bisphosphate

Hormonally Regulated

Regulation of GlycolysisPhosphorylation of Glucose Phosphorylation of Fructose-6-Phosphate Generation of Pyruvate

Pyruvate Kinase Feed-forward Regulation (+) F1,6,BP Large (-) G Essentially Irreversible Hormonally Regulated

PhosphoenolpyruvateADP

ATP

Pyruvate

Hormonal Regulation of GlycolysisInsulin Favors Glycolysis Upregulates the synthesis of Hexokinase Phosphofructokinase Pyruvate Kinase Glucagon Opposes Glycolysis Downregulates the synthesis of Hexokinase Phosphofructokinase Pyruvate Kinase

Glycolysis Takeaway PointsAnaerobic process Occurs exclusively in the cytosol Glucose (a six-carbon molecule) is degraded to 2 molecules of pyruvate (a three carbon molecule) Important sites of regulation phosphorylation of glucose phosphorylation of fructose-6-phosphate generation of pyruvate Energy Produced Per Glucose Molecule 2 NADH 2 ATP hexokinase PFK-1 Pyruvate Kinase

Fates of Pyruvate

Anaerobic Cytosolic


Anaerobic Conditions

Pyruvate is converted to lactate In the absence of oxygen, glycolysis is the only means of generating ATP. Regenerates the NAD+ required by glyceraldehyde-3-phosphate dehydrogenase during the payoff phase of glycolysis Anaerobic bacteria also rely on this pathway to maintain ATP production

Lehninger Figure p538

Aerobic Conditions


TCA Cycle

Pyruvate Dehydrogenase


Converts pyruvate to CO2 and Acetyl CoA TCA cycle Multi-subunit enzyme requires several cofactors A Thiamine pyrophosphate Coenzyme NAD+ FAD Lipoic Acid

Energy is harnessed in the form of NADH Negative allosteric regulation by Acetyl CoA and NADH

(mitochondrial matrix)


-Ketoglutarate Dehydrogenase

Lehninger Figure page 610

Analogous to Pyruvate Dehydrogenase Multi-subunit enzyme requires several cofactorsA Thiamine pyrophosphate Coenzyme NAD+ FAD Lipoic Acid

Energy is harnessed in the form of NADH

TCA CycleOccurs in the mitochondrial matrix Citrate synthase is the rate-limiting step Sites of NADH synthesis Isocitrate dehydrogenase - Ketoglutarate dehydrogenase Malate dehydrogenase Site of FADH2 synthesis Succinate dehydrogenase (member of the ETC) Site of GTP synthesis Succinyl-CoA synthetase

Regulation of the TCA CycleNegative Regulators Indicative of high energy state ATP NADH Acetyl-CoA Citrate Succinyl-CoA Positive Regulators Indicative of low energy state AMP/ADP CoA NAD+ Ca2+Lehninger Figure 16-18

TCA CycleFor each Acetyl CoA 2 CO2 1GTP 3 NADH 1 FADH2

ETC


ATP Producedby both substrate level phosphorylation and oxidative phosphorylation

Energy Producing Reaction 3 NADH 3 NAD+ FADH2 GDP + Pi GTP

Number of ATP produced 9 2 112 ATP/ Acetyl CoA oxidized

Catabolism of MacromoleculesConverges on the formation of Acetyl CoA

Krebs Cycle Takeaway PointsWhen oxygen is available Pyruvate Acetyl CoA(not technically a part of the TCA cycle)

Occurs exclusively in the mitochondrial matrix None of the reaction intermediates are consumed For each Acetyl CoA oxidized to 2 CO2 3 NADH 1 FADH2 1 GTP Reduced equivalents will be passed to the ETC for ATP synthesis Cycle is inhibited by high-energy substrates

Biochemistry Review for the NBDE 2008November 7, 2008General Outline Carbohydrate Metabolism Review Oxidative Phosphorylation Insulin Signaling Glucagon SignalingContact Information: [email protected]

Glycolysis Takeaway PointsAnaerobic process Occurs exclusively in the cytosol Glucose (a six-carbon molecule) is degraded to 2 molecules of pyruvate (a three carbon molecule) Important sites of regulation phosphorylation of glucose phosphorylation of fructose-6-phosphate generation of pyruvate Energy Produced Per Glucose Molecule 2 NADH 2 ATP hexokinase PFK-1 Pyruvate Kinase

Aerobic Conditions


TCA Cycle

Pyruvate Dehydrogenase


Converts pyruvate to CO2 and Acetyl CoA TCA cycle Multi-subunit enzyme requires several cofactors A Thiamine pyrophosphate Coenzyme NAD+ FAD Lipoic Acid

Energy is harnessed in the form of NADH Negative allosteric regulation by Acetyl CoA and NADH

TCA CycleOccurs in the mitochondrial matrix Citrate synthase is the rate-limiting step Sites of NADH synthesis Isocitrate dehydrogenase - Ketoglutarate dehydrogenase Malate dehydrogenase Site of FADH2 synthesis Succinate dehydrogenase (member of the ETC) Site of GTP synthesis Succinyl-CoA synthetase

TCA CycleFor each Acetyl CoA 2 CO2 1GTP 3 NADH 1 FADH2

ETC


Electron Transfer and Oxidative PhosphorylationGlucose metabolism thus far: 4 ATP 10 NADH 2 FADH2 ETC oxidizes reduced cofactors ATP Occurs in the inner mitochondrial membrane Hydrogen ions are pumped to the intermembrane space gradient Hydrogen ions drive ATP synthesisfigure-16-1

http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etran.htm

Flow of Electrons Complex I Complex II Complex III Complex IV NADH dehydrogenase Succinate dehydrogenase Cytochrome bc1 complex Cytochrome oxidase (a+a3)

Coenzyme Q (CoQ) (also called ubiquinone) Cytochrome c (Cyt c) Complex V ATPase


H+

H+

H+

e-

e-

e-

eH 2O

2H++ O2NADH + H+ NAD+

NADH is oxidized by Complex I Electrons are passed down the chain H+ are pumped to the intermembrane space Only complexes I, III, and IV pump H+ Three H+ are pumped/NADH Oxygen is the terminal electron acceptor

H+

H+

e-

e-

eH 2O

http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etran.htm 2 2H++ O

FADH2+

FAD

FADH2 is oxidized by Complex II Complex II is Succinate Dehydrogenase Electrons are passed down the chain H+ are pumped to the intermembrane space Two H+ are pumped per FADH2


H+ ee-

H+ ee-

H+

H+

H+ATPase

http://www.dentistry.leeds.ac.uk/biochem/lecture/etran/etran.htmO2 2H++

H 2O

NADH + H+

NAD+

Electron transfers generates a H+ gradient The energy generated by this gradient drives the synthesis of ATP

ADP + Pi

ATP

1 NADH 3 ATP 1 FADH2 2 ATP

Oxidative Phosphorylation Takeaway Points The electron transport chain is located in the inner mitochondrial matrix. NADH and FADH2 derived from the catabolism of glucose (and other macromolecules) donate electrons to the chain Oxygen is the terminal electron acceptor Hydrogen ions are pumped to the intermembrane space electrical gradient and pH gradient The flow of H+ back into the mitochondrial matrix powers the synthesis of ATP by the ATPase

Insulin SignalingElevated blood glucose cells of the pancreas Insulin Insulin signaling increases expression of GLUT-4 receptors in insulin responsive tissues Skeletal muscle Glycogen Adipose tissue Triacylglycerol (TAG) Insulin promotes Glucose Uptake Glycolysis Glycogen Synthesis HMP shunt Triacylglycerol Synthesis Insulin signal Dephosphorylation of enzymes Enzymes activated by insulin are dephosphorylated in the active state

Structure of Insulin

Synthesized as Preproinsulin (RER) Loses its signal sequence to become Proinsulin Disulfide bonds link Chain A and Chain B (RER) Loses its C-peptide to become Insulin (Golgi)

Insulin ReceptorCell-surface receptor Contains intrinsic tyrosine kinase activity Autophosphorylates Activates kinases and phosphatases Insulins effects are mediated by dephosphorylationLehninger Figure 12-6

Enzymes activated by insulin are active in dephosphorylated form Glycogen Synthase

Glucagon cAMP Protein Kinase A ATP ADP

Insulin SignalingIncreases Glucose uptake Glycogen Synthesis Protein Synthesis Fat Synthesis Decreases Gluconeogenesis Glycogenolysis Lipolysis Activates/Deactivates EnzymesAlters Gene Expression Increases Glucose Transport

Glycogen Synthase a (active) Pi

P Glycogen Synthase b (inactive)

H 2O

Protein phosphatase

Insulin

InsulinActivates/Deactivates Enzymes

Alters Gene ExpressionIncreases Glucose Transport

InsulinActivates/Deactivates Enzymes Alters Gene Expression

Increases Glucose Transport

Lehninger Box 11-02 Figure 1

Insulin Responsive Tissues GLUT- 4 Surface Expression

InsulinActivates/Deactivates Enzymes Alters Gene Expression Increases Glucose Transport


Insulin Takeaway PointsPeptide hormone Receptor has intrinsic tyrosine kinase activity

Glucose uptake Glycogen Synthesis Protein Synthesis Fat Synthesis Gluconeogenesis Glycogenolysis LipolysisAlters enzyme activity Activates Glycogen Synthase Alters gene expression Upregulates genes of glycolysis Promotes GLUT-4 translocation skeletal muscle & adipose tissue

Glucagon Signaling

Low blood glucose cells of the pancreas Glucagon Promotes the mobilization of blood glucose glycogenolysis gluconeogenesis Glucagon promotes protein and/or fat catabolism Glucagon signal phosphorylation of enzymes Enzymes activated by glucagon are phosphorylated in the active state Actions of Glucagon can also be performed by Epinephrine

Glucagon/Epinephrine Signaling


Activates G protein Activates Adenylate Cyclase cAMP Activates cAMP dependent protein kinase, Protein Kinase A

Glucagon/Epinephrine Signaling Activates a G protein Heterotrimeric () Inactive (GDP ) Active GTPLehninger Figure 12-14

()

Activates Adenylate Cyclase

Glucagon/Epinephrine SignalingLeads to the phosphorylation of enzymes Phosphorylation activates glycogen phosphorylase a Glycogenolysis

Phosphorylation inactivates glycogen synthase Halts Glycogen SynthesisLehninger Figure-15-25

GlucagoncAMP

Glucagon/Epinephrine Signaling

Protein Kinase AATP ADP



Decreases Glycogen Synthesis

H 2O

Protein phosphatase

Increases Glycogenolysis Gluconeogenesis Ketogenesis Uptake of amino acids

Insulin

GlucagoncAMP Protein Kinase AATP ADP

Glycogen is DegradedGlycogen P Phosphorylase a (active)ADP ATP H 2O Pi Insulin Protein phosphatase

Glycogen Phosphorylase Kinase b (inactive)Pi

P Glycogen Phosphorylase Kinase a (active)H 2O

Protein phosphatase

Glycogen Phosphorylase b (inactive)

Insulin

Glucagon Takeaway PointsPeptide hormone Receptor is associated with G-protein Activates Adenylate Cyclase cAMP (the second messenger)

Glycogenolysis Gluconeogenesis Ketogenesis Uptake of amino acids GlycogenesiscAMP activates Protein Kinase A physiological response is due to the phosphorylation of enzymes Enzymes activated by Glucagon are active when phosphorylated Glycogen Phosphorylase Kinase Glycogen Phosphorylase

Biochemistry Review for the NBDE 2008November 12, 2008General Outline- 2nd hour Carbohydrate Metabolism Glycogen Synthesis Hexose Monophosphate Shunt Glucose Mobilization Glycogenolysis GluconeogenesisContact Information: [email protected]

Major Pathways of Glucose Utilization Insulin Favors Glucagon Opposes

Glycogen Synthesis

Skeletal muscle and liver take up glucose and store it as glycogen [(14) and (16)] Glycogen synthesis occurs during the well-fed state Insulin favors glycogen synthesis Glucagon opposes glycogen synthesis

Glycogen Synthesisglucose 6- phosphate glucose 1- phosphatephosphoglucomutase

glucose 1- phosphate + UTP UDP-glucose + PPiUDP-glucose pyrophosphorylase

UDP- glucose is the substrate for glycogen synthesis Anomeric carbon of sugar is activated by attachment to UDP

Insulin favors glycogen synthesisLehninger Page 565

Glucagon opposes glycogen synthesis

Glycogen Synthesis Glycogen Synthesis

+ +



Glycogen SynthesisTwo enzymes glycogen synthase glycogen-branching enzyme

Synthase activated by insulin inactivated by glucagon

GlucagoncAMP

Glucagon Signals through the activation of Protein Kinase A Glycogen Synthase is inactive in the phosphorylated form




H 2O

Protein phosphatase

Insulin Signals through the activation of Protein Phosphatase Glycogen Synthase is active in the dephosphorylated form

Insulin

Glycogen Synthesis Takeaway PointsOccurs in skeletal muscle and liver [(14), (16)] Occurs during the well-fed state Substrate is UDP-glucose added to the non-reducing end of a growing glycogen chain Important enzymes Glycogen Synthase Glycogen Branching Enzyme Glycogen synthase is active in the dephosphorylated form (promoted by insulin) Glycogen synthase is inactive in the phosphorylated form (promoted by glucagon)

Insulin favors glycogen synthesis Glucagon opposes glycogen synthesis

Major Pathways of Glucose Utilization Insulin Favors Glucagon Opposes

Pentose Phosphate PathwayAlso referred to as Hexose Monophosphate Shunt 6-phosphogluconate Pathway Products Ribose 5-Phosphate NADPH Site of Regulation Glucose 6-Phosphate Dehydrogenase (G6PD)


Oxidative Phase of Pentose Phosphate PathwayGlucose 6-phosphateGlucose 6-Phosphate Dehydrogenase NADP+

NADPH

6-Phosphogluconate6-Phosphogluconate Dehydrogenase NADP+

NADPH

Ribose 5-phosphate + CO2

Products of the Pentose Phosphate Pathway NADPH Fatty Acid and Cholesterol Synthesis Counter Free Radicals RBCs are exposed to oxygen generated free radicals Glutathione functions to eliminate these free radicals NADPH is required to keep glutathione in the reduced state G6PD deficiency free radical damage hemolytic anemiaLehninger Box-14-03

Nonoxidative Phase of Pentose Phosphate Pathway


Occurs in cells not needing the pentose sugars Functions to regenerate glucose 6-phosphate

Glucose Utilization Take Away PointsGlucose is the bodys preferred substrate Insulin levels increase in response to elevated blood glucose Increase glucose uptake by skeletal muscle and adipose tissue Stimulates the phosphorylation of glucose GlucoseATP Hexokinase ADP

Glucose 6-Phosphate Glucose 6-Phosphate can be used in Glycolysis Glycogen Synthesis HMP Shunt

General Outline- 2nd hour Carbohydrate Metabolism Glycogen Synthesis Hexose Monophosphate Shunt Glucose Mobilization Glycogenolysis Gluconeogenesis

Maintenance of Blood GlucoseBlood glucose levels drop quickly after a meal

Glucagon:Insulin RatioActivates two processes to maintain blood glucose Glycogenolysis skeletal muscle liver Gluconeogenesis liver kidney

GlycogenolysisGlycogenolysis functions during the first 24 hours of fasting Glucose from liver glycogen blood glucose Glucose from muscle glycogen energy for the myocyte

Glucagon favors glycogenolysis Insulin opposes glycogenolysis

Glycogenolysis


Glucagon Activates

Insulin Inactivates

Glycogenolysis

Insulin

Glucagon activates Phosphorylase a by phosphorylating it [activates Phosphorylase b Kinase]


Insulin inactivates Phosphorylase a by dephosphorylating it [activates phosphorylase a phosphatase]

GlucagoncAMP Protein Kinase AATP ADP

Glycogen is DegradedGlycogen P Phosphorylase a (active)ADP ATP H 2O Pi Insulin Protein phosphatase

Glycogen Phosphorylase Kinase b (inactive)Pi

P Glycogen Phosphorylase Kinase a (active)H 2O

Protein phosphatase

Glycogen Phosphorylase b (inactive)

Insulin

GlycogenolysisGlucose 1-phosphate>>GlucoseActivated by Glucagon

Glucose 1-Phosphate Glucose 6-Phosphate Glucose 6-phosphatase

only in the liver and kidneyGlucose from liver glycogen blood glucose Glucose from muscle glycogen energy for the myocyte Lehninger Figure 15-4

Glycogenolysis Take Away Points

Glycogen is mobilized within four hours of eating Stimulated by Glucagon phosphorylates and activates glycogen phosphorylase kinase glycogen phosphorylase phosphorylates and inactivates glycogen synthase Occurs in liver blood glucose and muscle energy

Maintenance of Blood GlucoseBlood glucose levels drop quickly after a meal

Glucagon:Insulin RatioActivates two processes to maintain blood glucose Glycogenolysis skeletal muscle liver

Gluconeogenesis liver kidney

GluconeogenesisThe de novo synthesis of glucose Occurs in the liver>>kidney during fasting Substrates include all intermediates of glycolysis all intermediates of the TCA cycle lactate glycerol -ketoacids of glucogenic amino acids Is NOT a reversal of Glycolysis Must bypass Hexokinase Phosphofructokinase Pyruvate Kinase

Acetyl CoA is NOT a substrate for gluconeogenesis

Irreversible Steps in Glycolysis

Three important sites of regulation Large G Reactions are Irreversible

Gluconeogenesis in NOT a Reversal of Glycolysis

To counter the irreversible steps of glycolysis Four enzymatic steps Four ATP and Two GTP

Enzymes in GlycolysisHexokinase

Enzymes in GluconeogenesisGlucose-6-Phosphatase Fructose 1,6-bisphophatase Phosphoenolpyruvate Carboxykinase Pyruvate Carboxylase

Phosphofructokinase

Pyruvate Kinase

Conversion of Pyruvate to PhosphoenolpyruvateRequires Two Enzymes Pyruvate Carboxylase Phosphoenolpyruvate (PEP) CarboxykinasePyruvate Carboxylase (mitochondrial matrix) Pyruvate (3C) is Carboxylated to Oxaloacetate (4C) Requires Biotin, ATP, and CO2 Oxaloacetate can not cross the mitochondrial membrane so it is converted to malate PEP Carboxykinase (cytosol) Oxaloacetate (4C) is decarboxylated and phosphorylated to form PEP (3C) Requires GTP

MitochondrionPyruvate + CO2ATPAcetyl CoA

ADP + Pi

Pyruvate Carboxylase + Biotin

Oxaloacetate

Malate

Cytosol

Malate

OxaloacetateGTP PEP Carboxykinase GDP

Phosphoenolpyruvate + CO2

PEP

Reverse Glycolysis

Fructose 6-Phosphate

Conversion of Fructose 1,6-bisphosphate to Fructose 6-PhosphatePEP Reverse Glycolysis Fructose 6-Phosphate

Fructose 1,6- bisphosphateH 2O Fructose 1,6bisphosphatase P

Bypasses the irreversible phosphofructokinase-1 reaction Stimulated by High ATP Low AMP Inhibited by Elevated AMP Fructose 2,6-bisphosphate

Fructose 6 - phosphate

Conversion of Glucose 6-Phosphate to GlucoseFructose 6-Phosphate Glucose 6-Phosphate Glucose

Bypasses the irreversible hexokinase reaction Only occurs in the LIVER and KIDNEY Glucose 6-phosphatase is essential for glucose to be released into the blood stream Without this enzyme, glucose generated by glycogenolysis and gluconeogenesis can not be released to the body

Substrates for Gluconeogenesis All intermediates of glycolysis All intermediates of the TCA cycle Lactate Glycerol -Ketoacids of glucogenic amino acidsWhen glucose is limiting Muscles go into anaerobic metabolism Lactate Fats are catabolized Glycerol and fatty acids Proteins are catabolized amino acids

Acetyl CoA is NOT a substrate for gluconeogenesis

glucose lactate glucose

Cori CycleActive muscle mobilizes glycogen stores glucose Glucose is metabolized by glycolysis under anaerobic conditions lactate Lactate enters blood liver Gluconeogenesis resynthesizes glucose from lactate Glucose enters blood muscle Lehninger Figure 23-18 Gluconeogenesis occurs in LIVER and KIDNEY Glucose can be metabolized or stored as muscle glycogen

Substrates for Gluconeogenesis

Lysine and Leucine are NOT glucogenic

Regulation of GluconeogenesisGlucagon Phosphorylates enzymes deactivation Pyruvate Kinase PFK-2 Induces the expression of PEP carboxykinase Substrate Availability Glucogenic amino acids increase rate Reciprocal Control + Acetyl CoA Activates pyruvate carboxylase Inhibits pyruvate dehydrogenase Inhibits Fructose 1,6-bisphosphatase - AMP Activates PFK-1

Gluconeogenesis Takeaway PointsOccurs in liver and kidney Is NOT a reversal of Glycolysis Unique Enzymes Glucose 6-Phosphatase Fructose 1,6-Bisphosphatase PEP Carboxykinase Pyruvate Carboxylase (Biotin) Substrates include Glycolysis and TCA intermediates Glucogenic amino acids Glycerol Lactate Cori Cycle Lactate returns to the liver glucose


General Outline- 2nd hour Lipid Structure and Function Cholesterol and Fatty Acids Compartmental Barrier Derivatives of Cholesterol and FAs Fatty Acid Synthesis

CholesterolAbundant in cell membrane Also functions as a precursor Bile acids Vitamin D Steroid hormones Transported in Lipoprotein Particles Most abundant in LDL>HDL>VLDL Found in the diet and synthesized in all tissues Especially liver, adrenal cortex and reproductive tissuesLehninger Figure-10-16

Rate limiting step is catalyzed by HMG-CoA reductase Synthesis requires NADPH

Fatty Acids (FA)Lehninger Figure-10-1

Amphipathic Hydrophobic AND Hydrophilic Saturated Unsaturated Bonds No Double Bonds One Double

>90% are esterified Cholesteryl esters Phospholipids Triacylglycerides (TAG) Saturated Unsaturated Free FAs circulate bound to ALBUMIN ( during fasting)

Function Oxidized for energy by most tissues (esp. liver and muscle) Structural roles (phospholipids & glycolipids) Precursor of prostaglandin Major Energy Store (TAG)

Unsaturated Saturated

Essential

Triacylglycerol (TAG)

May also be referred to as triglycerides Major energy store found in adipocytes Three fatty acids esterified to glycerol Neutral Fat When mobilized FA Acetyl CoA TCA Glycerol glycolysis or gluconeogenesisLehninger Figure-10-2

Cholesterol and Fatty Acids From Diet or Synthesized de novo

Lehninger Figure-17-1

Chylomicrons

Largest, least dense lipoprotein Predominantly dietary triacylglycerol Generated in enterocytes lymph blood stream tissues Tissues will consume or store the dietary triacylglycerol Remnants return to the liver release their cholesterol degraded in lysosomes



Lipoprotein ParticlesChylomicrons Transport TAG from the diet to the tissues Apo B-48 LDL Particles Transport cholesterol to the tissues Receptor-mediated endocytosis Apo B-100 VLDL Particles Transport TAG synthesized by the liver to the tissues HDL Particles Transport cholesterol to the liver for elimination

Fatty Acid and Lipoprotein Particle Take Away PointsFatty acids part of phospholipids and glycolipids stored as neutral fat (TAG) free fatty acids are transported bound to albumin From the diet or synthesized from excess carbohydrates and proteins in the diet Saturated no double bonds Unsaturated double bonds palmitoleic, oleic linoleic, linolenic, arachidonic acid Chylomicrons VLDL LDL HDL Dietary TAG to the tissues Hepatic TAG to the tissues Cholesterol to the tissues Cholesterol to the liver for elimination


Glycerophospholipids Lecithin Sphingolipids Sphingomyelin

GlycerophospholipidsGeneral Structure Glycerol backbone Two Fatty Acids Phosphate Group + Polar Head Group

Know Lecithin (Phosphatidylcholine) glycerol phorphoric acid fatty acids choline(one of the most abundant phospholipids in cells)Lehninger Figure-10-8

Sphingolipids

General Structure Sphingosine backbone One Fatty Acid Head Group Sphingomyelin Sphingolipid prevalent in myelin Also contains choline Deficiency in degradation SphingolipidosesLehninger Figure-10-12

SphingolipidosesTay-Sachs Hexosaminidase A deficiency GM2 gangliosides accumulate Neurodegeneration Cherry-red Macula Gauchers Glucocerebrosidase deficiency Glucocerbrosides accumulate Hepatosplenomegaly Osteoporosis of long bones Niemann-Pick disease Sphingomyelinase deficiency Spingomyelin accumulation Hepatosplenomegaly Neurodegenerative

Lehninger Box 10-2 Figure-1

Phospholipid Membrane as a BarrierSmall, non-polar molecules enter cells by Simple Diffusion Down gradient without a transporterSTEROID HORMONES Circulate bound to a protein Diffuse through the membrane Have an INTRAcellular receptor (cytosolic or nuclear) Ultimately reach the nucleus Alters gene expression

Phospholipid Membrane as a BarrierLarge, polar substances require transporters to cross membranes Finite number of transporters saturation kinetics

Facilitated diffusion occurs down gradient does not require energy e.g. glucose uptake Active transport against gradient requires energy e.g. Na/K ATPase

Phospholipid Membrane Takeaway PointsAmphipathic Phospholipids and Glycolipids form lipid bilayer Know Lecithin and Sphingomyelin Cholesterol is abundant Membrane functions as a barrier polar substances require a transporter glucose or cell surface receptor peptide hormones nonpolar substances diffuse across membrane steroids

Three forms of transport simple diffusion facilitated diffusion require transporter active transport

down gradient

Steroid HormonesDerived from cholesterol Nonpolar, lipophilic Circulate bound to a carrier protein Enter cells by simple diffusion Bind to INTRAcellular receptors Ultimately reach the nucleus to alter gene expressionLehninger Figure-10-19

Response to steroid hormone stimulation is slow

EicosanoidsProstaglandins, Thromboxanes, LeukotrienesHave Hormone-Like Properties Physiological and Pathological Responses Vasoconstriction/Vasodilation Contraction/Relaxation of smooth muscle Bronchoconstriction and Anaphylaxis (Leukotrienes) Differences From Hormones Act locally Not stored Short half-life Produced by all tissues in small amounts Derived from arachidonic acid, a fatty acid component of the cell membrane


Arachidonic Acid liberated from phospholipid by Phospholipase A2 Converted by Cyclooxygenase to Prostaglandins and/or Thromboxanes Converted by Lipooxygenase to Leukotrienes

de novo Fatty Acids Synthesis

A large portion of fatty acids come from the diet chylomicrons tissues Fatty acids can also be synthesized from carbohydrates and proteins Primarily occurs in the liver and mammary glands, and, to a lesser extent, in adipose tissue Requires Acetyl CoA, ATP, Biotin, and NADPH Fatty Acid Synthesis Occurs in the Well-Fed State High Insulin:Glucagon Ratio

de novo Fatty Acids SynthesisStep One Transfer of Acetyl CoA across mitochondrial membraneThe Coenzyme A portion of the molecule can not cross the membrane Acetyl CoA condenses with Oxaloacetate to form Citrate Citrate crosses the membrane and is converted back to Acetyl CoA and Oxaloacetate

MitochondrionOxaloacetate + Acetyl CoACitrate Synthase

CoA

Citrate

CytosolCoA, ATP

CitrateATP-Citrate Lyase

ADP + Pi

Oxaloacetate + Acetyl CoA

de novo Fatty Acids SynthesisStep One Transfer of Acetyl CoA across mitochondrial membrane Step Two Carboxylation of Acetyl CoA to form Malonyl CoAOccurs in the cytosol Catalyzed by Acetyl CoA carboxylase Rate-Limiting Step Allosteric Regulation and Covalent Modification

Allosteric Regulation of Acetyl CoA CarboxylaseRate-limiting enzyme in de novo fatty acid synthesis Activated by Citrate, an indicator of high energy state Inhibited by Long-chain Fatty AcylCoA, the end-product Acetyl CoA + CO2ATP

LCFA-CoA

ADP + Pi

Acetyl CoA Carboxylase + Biotin

+

Citrate

Malonyl CoA

GlucagoncAMP

Covalent Modification of Acetyl CoA CarboxylaseGlucagon Signals through the activation of Protein Kinase A Acetyl CoA Carboxylase is inactive in the phosphorylated form Insulin Signals through the activation of Protein Phosphatase Acetyl CoA Carboxylase is active in the dephosphorylated form


Acetyl CoA Carboxylase (active) Pi

P Acetyl CoA Carboxylase (inactive)

H 2O

Protein phosphatase

Insulin

de novo Fatty Acids SynthesisStep One Transfer of Acetyl CoA across mitochondrial membrane Step Two Carboxylation of Acetyl CoA to form Malonyl CoA Step Three Elongation of fatty acid chain by Fatty Acid SynthaseUtilizes additional subunits of Acetyl CoA and NADPH

de novo Fatty Acids SynthesisStep One Transfer of Acetyl CoA across mitochondrial membrane Step Two Carboxylation of Acetyl CoA to form Malonyl CoA Step Three Elongation of fatty acid chain by Fatty Acid Synthase Step Four Esterification of FAs to Glycerol TAG

de novo Fatty Acids Synthesis Takeaway PointsFatty Acid Synthesis Occurs in the Well-Fed State High Insulin:Glucagon Ratio The rate limiting step is catalyzed by Acetyl CoA Carboxylase Allosteric regulation Covalent modification Predominantly occurs in the liver and mammary glands Requires Acetyl CoA, ATP, Biotin, and NADPH Occurs in the cytosol

Biochemistry Review for the NBDE 2008November 19, 2008General Outline- 1st hour Fatty Acid Catabolism Ketone Synthesis Protein Metabolism Production of Ammonia Urea Cycle Metabolism of -KetoacidsContact Information: [email protected]

EpinephrinecAMP

Fatty Acids Mobilization and OxidationTAGs are the bodys main energy store Concentrated energy source highly reduced and anhydrous To obtain energy TAG glycerol three free FAs Hormone-Sensitive Lipase Active when phosphorylated glucagon/epinephrine Inactive when dephosphorylated insulin


HormoneSensitive Lipase (inactive) Pi

Hormone- P Sensitive Lipase (active) H 2O

Protein phosphatase

Insulin

Triacylglycerol

HormoneSensitive Lipase

Fatty Acids Mobilization and Oxidation

Glycerol + Free Fatty Acids BLOODTissues

DHAP

Mitochondria

-Oxidation

Acetyl CoABrain and RBCs can NOT use FAs

TCA

-oxidation of Fatty AcidsOccurs in the Mitochondria Fatty Acids must use the Carnitine Shuttle Carnitine Acyl Transferase is inhibited by Malonyl CoA


-oxidation of Fatty AcidsOccurs during prolonged fasting Occurs in the mitochondria of liver and muscle Oxidizes fatty acids to Acetyl CoA FADH2 NADH TCA

ATPLehninger Figure-17-7

ETC

Fatty Acid Synthesis vs -Oxidation


Glucagon and Insulin also regulate Hormone-Sensitive Lipase Active when phosphorylated by Glucagon Inactive when dephosphorylated by Insulin

SynthesisGreatest flux through pathway Hormonal State favoring pathway Major tissue site Subcellular location Carriers of acyl groups Oxidation/reduction cofactors Two-carbon donor/ product Activator Inhibitor Product of Pathway After carbohydrate rich meal High Insulin:Glucagon Liver Cytosol Citrate NADPH Malonyl CoA (donates acyl) Citrate LCFA-CoA Palmitate

DegradationIn starvation Low Insulin:Glucagon Muscle, Liver Mitochondria Carnitine NAD+, FAD Acetyl CoA (product) Malonyl CoA Acetyl CoA

Well-Fed State Insulin SignalingAbundant Glucose Glycolysis Glycogen Synthesis HMP Shunt Excess Glucose and/or protein de novo Fatty Acid Synthesis

Fasting/Starvation Glucagon/Epinephrine SignalingLimited Glucose Glycogenolysis Gluconeogenesis Beta-oxidation of Fatty Acids Acetyl CoA Protein Catabolism

Ketone Bodies Acetoacetate, Acetone -hydroxybutyrateSynthesized in liver mitochondria HMG-CoA synthase is rate-limiting Acetoacetate and -hydroxybutyrate blood Acetyl CoA TCA Important sources of energy Soluble in aqueous environment Produced in excess of livers need Used in proportion to their concentration in the blood Ketone bodies are used by the brain RBCs can NOT use ketone bodiesLehninger Figure-17-18

Ketone Bodies Acetoacetate, Acetone -hydroxybutyrate

Formed during fasting and in Diabetes Type I Production in excess of rate of use Ketonemia Ketonuria

pKa value is approximately 4 Ketone dissociates, Release of hydrogen Lowers pH

Ketoacidosis

Fatty Acids Mobilization and Oxidation Takeaway PointsTriacylglycerol is the major form of energy storage Hormone sensitive lipase breaks TAG glycerol and FFAs Energy is derived from fatty acids by -oxidation Occurs in the mitochondria Degrades fatty acids to Acetyl CoA, FADH2, and NADH

-oxidation does NOT occur in the Brain or RBCsIn prolonged starvation Acetyl-CoA accumulates Ketones Rate-limiting step HMG CoA synthase Ketone Bodies= Acetoacetate, Acetone, -hydroxybutyrate

Brain can utilize Ketone Bodies RBCs can NOT use Ketone bodies

Protein MetabolismProtein catabolism occurs in three scenarios after a protein-rich meal during normal turnover of proteins during prolonged fasting or starvation Proteins catabolism results in the generation of free amino acids. Amino acids are degraded to carbon skeletons substrate for glucose ammonia toxic byproduct that is eliminated


Denaturation of Proteins


Loss of higher order structure No hydrolysis of peptide bonds

Digestive ProteasesStomach Parietal Cells HCl, Intrinsic Factor Chief Cells Pepsinogen Pepsin Pancreas Trypsinogen Chymotrypsinogen Procarboxypeptidases Intestine Enteropeptidase (Enterokinase) Cleaves trypsinogen to active trypsin Trypsin cleaves and activates itself and all other zymogens

Digestive Proteases

Trypsin (Serine Protease) Cleaves C-terminal to basic amino acids Chymotrypsin Cleaves C-terminal to aromatic amino acids Carboxypeptidases Remove the C-terminal residue of a peptide Aminopeptidases Remove the N-terminal residue of a peptide

Catabolism of Dietary Proteins

Amino acids are absorbed from the small intestine and enter the portal circulation The portal vein feeds the liver where amino acids are further catabolized Catabolism of amino acids requires two enzymes Transaminase (aminotransferase) Glutamate Dehydrogenase Amino acids are degraded to carbon skeletons (-ketoacids) ammonia (toxic byproduct)

AminotransferaseCatalyzes the first step in amino acid catabolism Transfers amino group from amino acids to -ketoglutarate Glutamate and the -keto-acid of the amino acid degraded are formed Requires pyridoxal phosphate as a cofactor (from Vitamin B6) Nearly all amino acids funnel their amino groups to glutamateLehninger Figure-18-4

Amino Acid + -Ketoglutarate

Glutamate + -Keto acid

Glutamate DehydrogenaseCatalyzes the second step in amino acid catabolism Only enzyme that can use either NAD or NADP Generates NH4+ and -Ketoglutarate


Glutamate

NH4+ + -Ketoglutarate

Degradation of Amino AcidsNH3 -NH2 of -amino acidsTransamination Aminotransferase

-Ketoglutarate

NADH (NADPH)

Oxidative Deamination Glutamate Dehydrogenase

-ketoacids

-NH2 of glutamate

NAD+ (NADP+)

NH3 is Toxic Must be incorporated into UREA for disposal

Alanine AminotransferaseAlanine -Ketoglutarate Alanine is deaminated to pyruvate Pyruvate is the -ketoacid of alanine

Pyruvate

Glutamate

Aspartate AminotransferaseOxaloacetate Glutamate Oxaloacetate is the -ketoacid of aspartate Formation of aspartate is favored during degradation Aspartate -Ketoglutarate Aspartate is required for the urea cycle

Protein Degradation Takeaway PointsDenatured in the stomach and degraded by specific proteases in the small intestine amino acids are absorbed Cellular proteins are continually synthesized and degraded (there is no storage form of protein). Degradation of amino acids proceeds through two steps Transamination (amino group is transferred to glutamate) Oxidative Deamination of Glutamate (generates free NH3 ) Ammonia is toxic and is detoxified in the liver Urea Cycle

Lehninger Figure18-10

The detoxification of ammonia occurs in the liver Glutamine is the predominant carrier of amino groups from most tissues Alanine transports amino groups from skeletal muscle and delivers its -ketoacid, pyruvate, to the liver. Glutamate NH3 and Aspartate

Urea Cycle


Occurs in both the mitochondria and cytosol Functions to convert ammonia to a non-toxic form Urea The two amino groups of urea are acquired from free ammonia (NH3) and aspartate The rate-limiting step Carbamoyl phosphate synthetase I

1. Ornithine transcarbamoylase 3. Argininosuccinase


2. Argininosuccinate synthetase 4. Arginase

The Urea Cycle and Citric Acid Cycle are Linked


Oxaloacetate from the TCA cycle is the alpha-ketoacid of aspartate Fumarate is generated in the urea cycle and returns to the mitochondrial matrix for the TCA

Flow of Nitrogen from Amino Acids to Urea

Amino acids are processed transamination oxidative deamination Amino groups are collected in the form of free ammonia and aspartate Free ammonia and aspartate donate their amino groups to the formation of non-toxic urea

Urea Cycle Takeaway PointsAmmonia is sent to the liver to be removed from the body as urea The two amino groups of Urea are derived from free NH3 and Aspartate The urea cycle occurs in the mitochondria and cytosol of the liver The cycle is tightly linked to the citric acid cycle Urea is released by arginase and enters the blood stream Urea is filtered and excreted by the kidney.

Disposal of Amino AcidsThe complete catabolism of amino acids (dietary or cellular) requires two reactions Transamination -Ketoacids Glutamate Oxidative Deamination of Glutamate -Ketoglutarate NH3

Amino Acid

NH3 + -Keto acidGlucose or Acetyl CoA

Amino Acid CatabolismThe -ketoacids of amino acids are converted to Acetyl CoA and/or intermediates of the TCA Amino acids degraded to Acetyl CoA are ketogenic Amino acids degraded to intermediates of the TCA are glucogenicLehninger Figure-18-15

Lysine and Leucine are purely Ketogenic

Degradation of Phenylalanine


Deficiency of Phenylalanine Hydroxylase or the cofactor tetrahydrobiopterin leads to Phenylketonuria

Phenylketonuria (PKU)Inability to hydroxylate phenylalanine to tyrosine Primarily caused by deficiency of phenylalanine hydroxylase Phenylalanine is degraded by an alternate pathway phenylketones Phenylketones in urine musty odor Symptoms MR, failure to walk or talk, seizures, hyperactivity Neonatal DiagnosisLehninger Figure-18-25

Treatment Dietary restriction Tyrosine becomes essential

In-born Errors of Amino Acid Catabolism

Amino Acid Catabolism Takeaway PointsDegradation of amino acids proceeds through two steps Transamination Oxidative Deamination of Glutamate NH3 (Urea Cycle) -keto acids (further degraded) Degradation of the carbon skeletons of amino acids ultimately lead to synthesis of glucose (glucogenic amino acids) ketone bodies (ketogenic amino acids) Lysine and leucine are entirely Ketogenic Deficiency of phenylalanine hydroxylase PKU

General Outline- 2nd hour Synthesis of Non-Essential Amino Acids Amino Acids as Substrates Water-Soluble Vitamins Fat Soluble Vitamins

Biosynthesis of Nonessential Amino AcidsEssential Amino AcidsCan not be synthesized in the human body. Must be acquired through the diet PVT TIM HALL (Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Lysine and Leucine; Histidine and Arginine are required for growth)

Nonessential Amino AcidsCan be synthesized in the human body. Substrates Intermediary metabolites (oxaloacetate aspartate) Essential amino acids (phenylalanine tyrosine) (methionine cysteine) Tyrosine and cysteine are conditionally non-essential

Amino Acid BiosynthesisSynthesized from intermediates of glycolysis, the citric acid cycle, and the HMP shunt Amino acids may serve as the precursor of other amino acids e.g. Serine glycine or cysteine Some amino acids are formed by the transamination of their -ketoacidsLehninger Figure 22-9

pyruvate alanine oxaloacetate aspartate -ketoglutarate glutamateNote: This figure includes essential amino acids that can not be synthesized by the human body

Synthesis of Amino AcidsNH3 -Ketoglutarate NADH (NADPH)

-NH2 of -amino acidsTransamination

Oxidative Deamination Glutamate Dehydrogenase

Aminotransferase

-ketoacids

-NH2 of glutamate

NAD+ (NADP+)

Transamination and oxidative deamination are readily reversible, according to the cells needs.

Amino Acids as SubstratesAmino acids are important precursors for the synthesis of specialized compounds in the human body Glycine is a precursor of the porphyrin ring of Heme oxygen binding in Hb substrate for bile pigments Several amino acids cooperate to synthesize compounds Creatine (an energy buffer in skeletal muscle) Glutathione (a critical reducing agent in RBCs) Nucleotide bases Several amino acids are decarboxylated to form special products Tryptophan Serotonin, Melatonin Tyrosine Dopamine, Norepinephrine, Epinephrine, Melanin Glutamate GABA Histidine Histamine

Synthesis of Catecholamines from TyrosineTyrosine hydroxylase catalyzes the first step. Dopa is the substrate for the neurotransmitters Dopamine Norepinephrine EpinephrineLehninger Figure 22-29

The aromatic amino acid decarboxylase requires pyridoxal phosphate as a cofactorTyrosine is also the precursor of Melanin Key enzyme: Tyrosinase

Synthesis of Serotonin from TryptophanTryptophan hydroxylase catalyzes the first step. The aromatic amino acid decarboxylase requires pyridoxal phosphate as a cofactor Pyridoxal phosphate is a cofactor in transamination reactions and decarboxylation reactions Serotonin functions in pain perception affective disorders regulation of sleep, temperature and blood pressure


Decarboxylation of Amino Acids


Histamine GABA Inhibitory Neurotransmitter Deficiency leads to seizures Potent Vasodilator Released from mast cells during allergic response Production in the stomach stimulates acid production

Amino Acids as Substrates Takeaway PointsAmino acids function as the precursors of other specialized compounds bile pigments nucleotides glutathione Specific Amino Acids and Their ProductsTyrosine Dopamine, Norepinephrine, Epinephrine, Melanin Tryptophan Serotonin, Melatonin Histidine Histamine Glutamate GABA

VITAMINS

Water-soluble

Fat-solubleVitamin A Vitamin D Vitamin E Vitamin K

NonB-complexAscorbic acid (Vitamin C)

B-complex

Energy-releasingThiamine (B1) Riboflavin (B2) Niacin (B3) Biotin Pantothenic acid

HematopoeiticFolic Acid Vitamin B12

OtherPyridoxine (B6) Pyridoxal Pyridoxamine

Ascorbic Acid (Vitamin C)Water-soluble vitamin Functions as an anti-oxidant Essential coenzyme in the hydroxylation of proline and lysine residues in collagen Required for normal connective tissue development and wound healing Vitamin C deficiency leads to Scurvy Sx sore, spongy gums loose teeth fragile blood vessels swollen joints anemia

Thiamine (Vitamin B1)Functions as Thiamine Pyrophosphate (TPP) Coenzyme in oxidative decarboxylations pyruvate dehydrogenase -ketoglutarate dehydrogenaseThiamine Deficiency Beriberi (B1B1) Common in diets consisting mainly of polished rice Sx tachycardia, vomiting convulsions, death Wernicke-Korsakoff Common in alcoholics Sx apathy, loss of memory rhythmic side-to-side movement of eyes

Riboflavin (Vitamin B2)Precursor of the cofactors FMN and FAD Critical for oxidation-reduction reactions Riboflavin Deficiency Symptoms include dermatitis, cheilosis and glossitis

Niacin (Vitamin B3)Precursor of the cofactors NAD+ and NADP+ Critical for oxidation-reduction reactions Tryptophan can compensate for niacin deficiency Niacin Deficiency = Pellagra Three Ds Dermatitis Diarrhea Dementia

BiotinCoenzyme in carboxylation reactions Required for pyruvate carboxylase acetyl CoA carboxylase Biotin Deficiency is Rare Pantothenic Acid Component of coenzyme A Required for TCA, fatty acid synthesis and degradation Pantothenate Deficiency is Poorly Characterized

FolateFound in leafy green vegetables Functions in one-carbon transfers as coenzyme tetrahydrofolate Important in the synthesis of amino acids, purines and thymine

Folic Acid DeficiencyMegaloblastic Anemia Failure to synthesize purine and thymidine nucleotides Accumulation of large, immature RBC precursors in the bone marrow Neural tube defects Spina bifida- failure to close the caudal portion Anencephaly- failure to close the rostral portion

Vitamin B12 (Cobalamin)Required for the synthesis of methionine and degradation of odd-numbered fatty acids Only synthesized by microorganisms Obtained from eating foods derived from animals Absorption B12 binds to intrinsic factor, produced by the parietal cells of the stomach. Intrinsic factor and B12 are absorbed in the ileum Most B12 deficiency is due to autoimmune destruction of the parietal cells Pernicious Anemia Megaloblastic anemia CNS defects (irreversible)

Pyridoxine (Vitamin B6)May be referred to as pyridoxine, pyridoxal, or pyridoxamine Converted to the biologically active form pyridoxal phosphate Is a cofactor for a variety of enzymes, especially those associated with amino acid catabolism

Reaction Type Transamination Deamination Decarboxylation

Example Oxaloacetate + Glutamate Aspartate + -ketoglutarate Serine Pyruvate + NH3 Histidine Histamine + CO2

VitaminFolic Acid Vitamin B12 Vitamin C Vitamin B6 Vitamin B1 Niacin Riboflavin Biotin Pantothenic Acid

FunctionTransfer of 1-carbon units Synthesis of purines and thymine Degradation of oddnumbered fatty acids Antioxidant Cofactor for Hydroxylations Cofactor for a.a. catabolism Cofactor for oxidative decarboxylation reactions Electron Transfer Electron Transfer Carboxylation reactions Acyl carrier

DeficiencyMegaloblastic Anemia Neural Tube Defects Megaloblastic Anemia Neuropsychiatric Sx Scurvy (loose teeth & Poor wound healing) Rare Beriberi Wernicke-Korsakoff Pellagra (3 Ds) Rare Rare Rare

Fat Soluble Vitamins ADEKAbsorbed from the diet in mixed micelles Pancreatic insufficiency can lead to steatorrhea and deficiency of fat-soluble vitamins (ADEK) Function much like steroid hormones readily diffuse through the lipid membrane have nuclear receptors alter gene expression Are stored in fat deposits in the human body. Excessive intake can lead to toxicity.

Vitamin A (Retinol)Retinoids class of compounds related to Vitamin A required for vision, growth and epithelial tissues Deficiency causes night blindness and xerophthalmia Excess Vitamin A leads to toxicity teratogenic

Vitamin EPrimarily functions as an antioxidant, preventing the nonenzymatic oxidation of compounds by molecular oxygen or free radicals Deficiency in adults is rare Least toxic of the fat-soluble vitamins (ADEK)

Vitamin DTwo precursors Ergocalciferol (Acquired through the diet) 7-Dehydrocholesterol (Synthesized in the skin) Precursors are activated by hydroxylation position 25- in the liver position 1- in the kidney Active 1,25 dihydroxycholecalciferol Synthesis of active Vitamin D is stimulated by parathyroid hormone in response to low blood calcium.


Vitamin D (contd)Vitamin D functions to maintain blood calcium levels increases calcium absorption in the intestine limiting loss of calcium through the kidney promoting bone resorption in conjunction with PTH Vitamin D Deficiency Rickets (in children) Collagen matrix of bone is formed but is not completely mineralized Soft, pliable bones Osteomalacia (in adults) Existing bones are demineralized and are much more susceptible to fracture Vitamin D deficiency can be a result of insufficient UV exposure dietary deficiency advanced renal disease

Vitamin KVitamin K is essential for post-translational modification of clotting factors II, VII, IX, and X Blood clotting factors are synthesized in the liver where they undergo Vitamin K-dependent carboxylation of their glutamate residues. Glutamate carboxylation is required for clotting factors to interact with activated platelets at the site of a wound. Modified glutamate has an overall charge of -2. Warfarin is a competitive inhibitor used as an anticoagulant in patients at risk of heart attack or stroke. Deficiency of Vitamin K is rare in adults microbes in gut synthesize Vitamin K

Blood Clotting CascadeDamage to the endothelium exposure of subendothelial cells Von Willebrand Factor and collagen recruit & activate platelets 1- expression of fibrinogen receptors 2- expression of phosphatidylserine phosphatidylserine binds Ca2+ positively charged calcium recruits the negatively charged clotting factors Intrinsic Pathway all the components required for clot formation are intrinsic to the blood Extrinsic Pathway- requires cooperation of tissue factor at the site of the wound for clot formation

Blood Clotting Cascade (contd)Intrinsic Pathway XI Exposed negative Extrinsic Pathwaysurface

XVIICells expose Tissue Factor

XIIa IXa+

XII

XIa

VIIa

IX

VIIIa

Fibrinogen Thrombin Fibrin

Prothrombin

Xa + Va

Factors II, VII, IX and X are Vitamin K dependent Factors V and VIII are coenzymes

Fat-Soluble Vitamins ADEK VitaminVitamin A

FunctionReproduction Vision Maintenance of epithelial tissue Calcium Uptake Antioxidant Cofactor for -Carboxylation of glutamate in clotting factors

DeficiencyImpotence Night Blindness Xerophthalmia Rickets (children) Osteomalacia (adults) Rare RareCompetitively inhibited by Warfarin

Vitamin D Vitamin E Vitamin K

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