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Control and Integration of Metabolism Arjumand Warsy BCH 540 (1425-26) Major Goals of Metabolism To provide precursors for synthesis of Macromolecules i.e. proteins, polysaccharides lipids, DNA, RNA, etc. To extract energy (ATP) and reducing power (NADPH, NADH) from nutrients, for synthesis of complex molecules A.Warsy Types of Metabolic Pathways Anabolic pathways Catabolic pathways • Synthesise larger molecules from small molecules • use energy NADPH, NADH • Degrade large molecules to smaller molecules. • Produce ATP, NADH, NADPH e.g. Glycolysis, fatty acid oxidation, a.a.oxidation Amphibolic pathways • Both catabolic and anabolic in nature e.g. Gluconeogenesis, Fatty acid synthesis, Glycogen synthesis, Lipid synthesis Protein synthesis ANABOLISM + CATABOLISM = A.Warsy e.g. T.C.A. cycle METABOLISM (Endothermic reactions) (Exothermic Reactions) Basal Metabolic Rate (BMR) Defination: Amount of energy required to maintain basic physiologic functions while at rest • Higher in males than females Calculation of BMR Men BMR (Kcal/hr/kg = 66 + (13.7 x W) + (5 x H) – (6.8 x A) Women: BMR (Kcal/hr/kg = 65.5 + (9.5 x W) + (1.8 x H) – (4.7 x A) BMR During pregnancy and Lactation 2nd and 3rd Trimester : Additional 300 Kcal/24 hrs A.Warsy Lactation : Additional 500 Kcal/24 hrs Energy from Main Constituents of Human Nutrition Macronutrient: Energy content Kcal/g - Carbohydrate …………………. - Protein …………………………. - Fat ……………………………… 4.1 4.1 9.3 Micronutrients: -Vitamins ……………………….. - Minerals ………………………. A.Warsy Nil Nil Integration of Catabolism and Anabolism by Energy Carriers Catabolic Pathway Anabolic Pathway Metabolic Fuels Cellular Components Carbohydrates Proteins Fats Polysaccharides Nucleic acids Proteins Lipids ADP + P NAD+, NADP+ ATP, NADH NADPH Energy carriers and reducing powers small energy-depleted metabolites (CO2, H2O, NH3) A.Warsy Precursor molecules (Monosaccharides, Amino acids, Fatty acids, Purines, Pyrimidines, etc. Overview of Metabolism A.Warsy A.Warsy Regulation of Metabolism Homeostatis is living organisms demands that metabolism be carefully controlled. Cells continuously adjust rate of metabolic pathways to ensure adequate energy and building blocks are available to carry out normal cellular functions. Several mechanisms exist to control metabolism through control of certain key enzymes, which play an essential role in control of metabolism as their concentration and activity may be altered. These are Regulatory Enzymes. A.Warsy Level of Control The regulation of a metabolic pathway may occur at several levels. Since enzymes are the indispensable catalysts for all reactions in the cell, the control of metabolism is ultimately concerned with the regulation of enzyme activity. An alteration in enzyme activity can be achieved in two fundamental ways: Coarse Control: control of the amount of an enzyme. A slow process as it involves protein synthesis. Fine Control: control of the activity of the enzyme. A fast process as it involves changing the activity of enzyme already available A.Warsy in the cells. Types of Regulatory Mechanisms Coarse Control Fine control Control of amount of an enzyme by ↑ or ↓ synthesis (Protein synthesis (inducible enzyme)) Control of Enzyme Activity By noncovalent interaction By covalent interaction By Hormones, Growth Factors Other molecules Substrate Availability e.g. ↑Substate →↑ product ↑ Glucose → ↑ Glycogen ↑ FA + Glycerol.3.p →↑ TG ↑ AA+ ↑ energy → ↑ protein Allosteric Regulation e.g. ↑ ATP→inhibition of PFK and hence glycolysis (Feedback inhibition A.Warsy Protein-protein interaction e.g. ↑activity of many regulatory proteins by binding to calmodulin ↑(Ca++ binding protein) Coarse Control •In this central process a metabolite or other controlling substance causes an increase or decrease in the amount of enzyme (protein) synthesized. •This takes a long time to be completed as it involves the couple machinery of protein synthesis: – - Transcription – - Translation A.Warsy Fine Control • In this process a metabolite causes increase or decrease in the activity of the pre-existing enzyme. • This is a rapid control and is easily switched ‘On or Off’. • Several factors affect the enzyme activity: - S availability - Cofactor availability - Product removal - Feedback inhibition - Compartmentation - Covalent modification of enzyme activity - Hormonal control A.Warsy (a) Substrate Availability • Any metabolic pathway could be regulated by availability of substrate. • A reduction in substrate conc. will decrease the activity of a enzyme (provided it is not saturated with substrate) and this could result in a decreased flux through the pathway. • An increase in substrate concentration could stimulate the pathway. • For some metabolites such as blood glucose and intracellular glycogen several factors play a part in order to regulate their concentration. • The concentration of plasma fatty acids appears to play a fundamentally important role in the regulation of their oxidation by various tissues, and in turn their oxidation can modify the rate of carbohydrate utilization by the animals. In such situations, if the substrate conc. is shown to be regulatory the emphasis must also lie on the factors that are responsible for A.Warsy changes in substrate conc. e.g. conc. of plasma FA seems to play a role in the regulation of their oxidation. • Other factors play a part in control of plasma FFA conc. e.g. plasma FA conc. ↑ (due to lipolysis) during: • (a) Stress (caused due to relese of adrenaline and noradrenaline) due to stimulation of adenyl cyclase ∴ ↑ in cAMP in adipose tissue ∴ causing ↑ in release of FFA from adipose tissue ∴ ↑ in plasma FFA TAG → FFA + Glycerol (b) Starvation causes ↑ in FFA in serum, due to decreased esterification of FFA. So FFA + Glycerol ↓→ TAG and also TAG → FAA + Glycerol ↑. Several hormones may be involved in this during starvation e.g. Growth hormone stimulates lipolysis. Lack of Insulin : During starvation there seem to be fact in plasma insulin levels and so the lipolysis hormones causes lipolysis (as insulin A.Warsy inhibits lipolysis). Conti.. • (d) Glucagon simulator lipolysis (due to glucagon ↑ in fasting subjects). • (e) Other factors: e.g. TSH, ACTH is also elevation plasma FFA levels. • (f) Prolonged muscular activity alters FFA levels. • ↑ FFA due to mobilization of FFA. • ↑ in concentration causes ↑ in FFA oxidation by various tissues (so a good fuel alternate + glucose) and so glucose utilization ↓ in muscles and possibly other tissues). ↑ oxidation of FFA, ↑ in KB in liver. A.Warsy (b). Cofactors Availability • Cofactors play important part in control of a pathway. Inhibition of enzyme activity can be achieved by ↓ the concentration of its cofactor e.g. FA oxidation can be controlled by the concentration of carnitine. • So theoretically it is possible that the concentration of carnitine could regulate the rate of FA oxidation. • Another example of the control by cofactor availability is the regulation of Electron transport chain and oxidative phosphorylation in the introduction and presence of adenine nucleotides is necessary for synthesis of ATP. ↓ ADP causes ↓ in the oxidation phosphorylator and ↓ in ATP causes an increase. • ADP ATP A.Warsy Examples of cofactors • Availability of Ca++ for: – blood clotting – Muscle contraction – Transmission of nervous impulses • Availability of Mg++ for: – Replication – Transcription – Translation – Activity of all kinases • Availability of ATP or ADP A.Warsy • Availability of carnitine in lipid metabolism Role of Ca++ in muscle contraction • In muscle: ATPase ATP ADP (Regulated by Ca++ ) • Absence of Ca ++ in the sarcoplasmic space during resting stage decreases enzyme inactive- no muscle contraction. • Muscle stimulation → Ca++ is released from sarcoplasmic reticulum and activates ATPase. So ATP is broken down and muscle contraction occurs Ca++ pump then removes the Ca++ and enzymes become inactive and resting A.Warsy state is stored. A.Warsy A.Warsy (c) Product Removal • The removal of a product of metabolic pathway can control the rate of its formation from the substrate. This is not so important for the major metabolic pathways but does play part in some of the minor pathway. e.g. pyruvate → lactate in muscle goes to lactate in blood. As the product is removed from muscle more pyruvate could change to lactate. A.Warsy (d) Regulatory enzymes • Some enzymes possess properties that specifically endow them with regulatory roles in metabolism. Such more highly specialized forms are called Regulatory enzymes. Two types of regulatory enzymes: • (a) Allosteric enzymes: Whose catalytic activity is modulated through the non-covalent binding of a specific metabolite at a site as the protein other than the catalytic site, and • (b) Covalently modulated enzymes, which are interconverted between active and inactive forms by the action of other enzymes. Some of the enzymes in the second class also respond to noncovalent allosteric modulators. A.Warsy Regulation of metabolism by regulatory enzymes • These two types of regulatory enzymes are responsive to alterations in the metabolic state of a cell or tissue on a relatively short time– allosteric enzymes within seconds and – covalently regulated enzymes within minutes. A.Warsy Allosteric Enzymes Activity of these enzymes may change in response to small changes in environment. These enzymes catalyse Rate Limiting Step or Committed step i.e. once this step occurs the whole pathway must go on. They are Feed back inhibited by the end product. These are allosteric enzymes. Branched reactions Multiple feedback loops provide addition control. E F↔G↔H E E A→B→C↔D↔E Z E X ↔ Y ↔A.Warsy Allosteric Enzymes • Control [regulatory] enzymes • Have quaternary structure • Have active site and modulatory site – Active site binds substrate to give product – Modulatory site binds +ve or – ve modulator to increase or decrease the activity of the active site • Catalyse an irreversible reaction • Inhibited by end product A B C D E F G H Feedback inhibition • Activated by substrate and other positive modulators • Do not obey Michaelis Menten Kinetics A.Warsy Heterotropic and Homotropic Enzymes • Heterotropic and homotropic effect depends on the nature of the modulating molecules. • Heterotropic enzymes are stimulated or inhibited by an effector or modulator molecule other than their substrate e.g. modulator molecule other than their substrate e.g. threonine dehydratase, the substrate is threonine and the modulator is L-isolecuien. • In Homotropic enzymes, the substrate also functions as the modulator. Homotropic enzymes contain two or more binding sites for the substrate- Modulation of these enzymes depends on how many of the substrate sites are occupied. Most allosteric enzymes are of the mixed homotropic-heterotropic types in which both the substrate and some other metabolite A.Warsy may function as modulators. Kinetics of Allosteric Enzymes • Allosteric enzyme do not show classical Michaelis Menten Kinetics between substrate concentration. (I.e.Vmax and Km). • Their kinetic properties are greatly altered by variations in the concentration of the allosteric modulator. • Many allosteric enzymes particularly homotropic show a sigmoid curve relating initial velocity to substrate concentration rather than the rectangular hyperbolic curve. • Sigmoid curve implies that binding of the first substrate molecule to the E enhance the binding of subsequent substrate molecules to the other substrate site. I.e. Co-operative effect A.Warsy A.Warsy • The activity of allosteric enzymes is modulated by low MW allosteric effectors (modulators), which generally have no or very little structural similarity to the substrate or coenzymes for the regulatory enzymes. • There are two types of modulators: – Negative modulators – Positive modulators • Negative allosteric effector (feedback inhibitor) binds to a allosteric site on the E and inhibits. • Positive allosteric effector activates the activity of the enzyme. A.Warsy Positive and negative modulator A.Warsy Feedback Regulation • Feedback regulation of enzyme activity is the most flexible and biologically widespread mechanism of metabolic control. • Feedback control in metabolic systems operates solely through the regulation of the activity of enzymes their catalyse non-equilibrium reaction. A→B↔C ↔D ↔E ↔F ↔G ↔H ↔I ↔J E Feedback Inhibition • The regulation of a pathway by control of the activity of regulatory enzymes that catalyse non-equilibrium reaction is of great significance. A.Warsy A.Warsy Structure of PFK A.Warsy Activation of PFK by ADP A.Warsy A.Warsy Pyruvate dehydrogenase complex Control of TCA cycle A.Warsy Steps in Glycolysis A.Warsy Contd… A.Warsy Regulation of Glycolysis DHAP Hexokinase* Glucokinase Glucose Inhibited by Glucose-6phosphate Phosphofructokinase (PFK)* G6P ↔F6P G3p F1,6.p2 Activated by AMP & Fructose 2,6, diphosphate Inhibited by ATP & Citrate 1,3,BPG ↔3 PG 2 PG Pyruvate PEP kinase* Activated by F1, diphosphate inhibited by ATP & pyruvate alanine *Allosteric Enzyme (Regulatory) catalyse irreversible reactions A.Warsy Formation of lactate from Pyruvate ↓O2 Pyruvate dehydrogenase complex ↑O2 A.Warsy Acetyl Co A A.Warsy A.Warsy Entry of Glycogen, Starch, Disaccharides & Hexoses into the Pathway of Glycolysis Lactose Lactase Trehalose D-Galactose Glycogen, starch Trehalase D-Glucose Phosphorylase(p) ATP Sucrose Sucrose D-Fructose Fructokinase G.I.P. mutase HK ATP ATP HK Fructose-1-P Fructose-1-P aldolase Glyceraldehyde * Dihydroxy acetone P Triose kinase UDP-Gal UDP-Glu Manoase Glucose-6 P ATP HK Mannose-6-P Fructose-6 P Triose phosphate isomerase A.Warsy Fructose 1,6 dip Phospho-mannose Isomerase Glyceraldehyde 3-P Pyruvate Lactic Acidosis and Pyruvate Dehydrogenase (PDH) Deficiency Def. of PDH (Acquired or inherited) Lactic acid in blood Lactic acidosis Acquired in: Chronic alcoholism and Thiamine deficiency Arsenate and mercurial poisoning Pyruvate kinase deficiency – inherited. Haemolytic anaemia due to insufficient glycolysis and hence ↓ ATP in RBCs. Hexokinase deficiency – Inherited A.Warsy Hemolytic anaemia due to ↓ ATP in RBCs. Tricarboxylic Acid Cycle Catabolism of acetyl CoA → 2CO2 + 3NADH FADH2 +GTP Amphibolic pathway Anabolic – Synthesis of a.a., glucose; fatty acids Heme from Intermediates of TCA cycle Deficiency of vitamins: Niacin, Thiamine, Panthothenic acid, Riboflavin, result in decrease oxidation of Acetyl CoA and ↑ formation of lactic acid ∴ Lactic acidosis. A.Warsy A.Warsy A.Warsy A.Warsy The electron transport chain A.Warsy A.Warsy Pentose Phosphate Pathway (Hexose Monophosphate Shunt) <10% glucose oxidised by PPP. Glucose converted to riboses with production of NADPH. (C6) (C5) Function: • Synthesis of riboses for nucleic acid synthesis. • Synthesis of NADPH for biosynthesis of FA, cholesterol etc. Glucose H-K G-6-P NADP+ G-6-PD NADPH + H+ 6-phosphogluconate CO2 Ribulose-5-P C3 C4 C5 C6 C7 F6P Intermediates PPP • No ATP used or produced. • Occurs in all cells. Rich in liver, adipose, lactating mammary glands • NADPH protects the red cells from oxidative stress of drugs, metabolites. Glucose-6-phosphate dehydrogenase deficiency • Most common enzymopathy. • XR • Hemolytic anaemia under oxidative stress. A.Warsy (Favism), Drugs e.g. sulfonamides antimalarial drugs; nitrofurans. A.Warsy Covalent Modification • Covalently modulated enzymes, are interconverted between active and inactive forms by the action of other enzymes. Some of the enzymes in the second class also respond to noncovalent allosteric modulators. • These two types of regulatory enzymes are responsive to alterations in the metabolic state of a cell or tissue on a relatively short timeallosteric enzymes within seconds and covalently regulated enzymes within minutes. A.Warsy Types of covalent modifications By covalently modifying enzymes and hence its activity By covalent interaction Phosphorylation and dephosphorylation protein Protein kinase O E-O-P-O O E Protein phosphatase Limited Proteolysis e.g. ↑ activity • Glycogen phosphorylase • Lipase ↓ Activity • Glycogen synthetase • Lipid synthetase Protease Zymogen (inactive) Active E + Peptide e.g. Prothrombin Trypsinogen Proelastase A.Warsy Thrombin Trypsin Elastase A.Warsy A.Warsy A.Warsy A.Warsy A.Warsy (e) Compartmentation: An Animal Cell A.Warsy • Eucaryotic cells have many compartments, separated from each other by a semi-permeable membrane. • Compartmentation plays an important role in control of metabolic activities. e.g: – lysosomes contain hydrolytic enzymes such as proteases, ribonucleases, phosphatases – Harmful for cell. But safely compartmented in lysozymes. Released under certain conditions so that they degrade macromolecules. • Compartmentation also makes it possible to have same processes going on at the same time in the same cell e.g. F.A. oxidation → Acetyl CoA in mitochondrial matrix F.A. synthesis from Acetyl CoA in cytoplasm. A.Warsy Pathways in different cell compartments Acetyl CoA A.Warsy Interconversion of Metabolic Fuel Storage: Liver Muscles Adipocytes Glycogen Proteins Triglycerides ↓ ↑ Amino Acids ↓ ↑ Fatty acids Glucose Amino Acids Fatty acids Glucose Amino Acids ↑ ↓ Glucose Transport: Tissue Metabolism Pyruvate Fatty Acids Acetyl CoA Cholesterol Lactate Ketoones Lactate CO2 Ketones ATP Synthesis A.Warsy