ADVANCED BIOCHEMISTRY BISC 6310 FALL IUG, Fall 2013 Dr. Tarek Zaida C OURSE TOPICS : T EXTBOOK : B IOCHEMISTRY, 5 TH ED. (L UBERT S TRYER ) 1.Basic concepts of metabolism (ch. 8 & 14) 2.Glycolysis and gluconeogenesis (ch. 16) 3.The citric acid cycle (ch.17) 4.Oxidative phosphorylation (ch. 18) 5.Glycogen metabolism (ch. 21) 6.Fatty acids metabolism (ch. 22) 2 7. Protein turnover and amino acids metabolism (ch. 23) 8. Nucleotide biosynthesis (ch.25) 9. Biosynthesis of membrane lipids & steroids (ch. 26) 10. The Integration of metabolism (ch. 30) 11. Signal Transduction (ch.15) 3 A CTIVITIES AND EXAMS Activities: At the end of the term every student will present an oral presentation of his/her choosing, covering one of the medical-related disorders connected to any of the course chapters You have to let me know which topic you have chosen within 1 week from now, so that I may grant you the permission to proceed with your research. 4 The evaluation of the presentation will be based on: Performance (oral) Content (oral & written) Time frame (limited to 10 min.) Note: Be ready to give me up your presentation electronically or viaat : or The time of the presentation should not exceed 10 minutes. Exams: Two exams will be held in the middle and at the end of the semester. The material of the final exam will include up to 20% of the chapters of the first term. 5 You will be given 4 Quizzes., the first, and second before midterm & the third and fourth before final exam. Making-ups are not allowed neither for presentations nor for quizzes nor for midterm (STRICTLY ENFORCED) Class attendance is strongly recommended, But once you decide not to come to class, its your responsibility to keep yourself posted and updated with what has been done/ said during class. I will always welcome your suggestions as it is the best way to improve the outcome of this class (you can talk to me in person or if you feel more comfortable drop me an) Office hours: male students Tue 2-3 pm female students Sun: 2-3 pm 6 G RADING : Points 1Presentation7 2Different Electronic Tools 7 3Quizzes16 4Midterm exam20 5Final exam Total METABOLISM BASIC CONCEPTS & DESIGN The quantitative study of cellular energy transductions and the chemical reactions underlying these transductions. 8 E NZYMES Enzymes are the most sophisticated biological catalysts known. catalysts alter the rates of chemical reactions but are neither formed nor consumed during the reactions they catalyze. Most enzymes are proteins. Some nucleic acids exhibit enzymatic activities (e.g., rRNA). 9 E NZYME C HARACTERISTICS : R ATE E NHANCEMENT Enzymes significantly enhance the rates of reactions: 10 E NZYME C HARACTERISTICS : T URNOVER N UMBERS The turnover number is used to rate the effeciency of an enzyme. It tells how many molecules of reactant a molecule of enzyme can convert to product(s) per second. How fast can an enzyme produce products? 11 E NZYME C HARACTERISTICS : S PECIFICITY Enzymes can be very specific. For example, proteolytic enzymes help hydrolyze peptide bonds in proteins. Trypsin is rather specific Thrombin is very specific 12 The specificity of an enzyme is due to the precise interaction of substrate with the enzyme. This is a result of the intricate three-dimensional structure of the enzyme protein. E NZYME C HARACTERISTICS : R EGULATION Enzymes can enhance the rates of reactions by many orders of magnitude. A rate enhancement of means that what would occur in 1 second with an enzymes help, would otherwise require 31,710,000,000 years to take place. Thus, regulation of enzymatic activity is in a sense, regulation of metabolism, or any other cellular process 13 E NZYME C OFACTORS Many enzymes use same cofactor Cofactors are split into two groups: Metals Coenzymes (small organic molecules) Most vitamins are coenzymes. When tightly bound to enzyme, cofactor =prosthetic group 14 Apoenzyme + cofactor = Holoenzyme E NZYMES G IBBS F REE E NERGY Gibbs Free Energy, G, determines the spontaneity of a reaction: G must be negative for a reaction to occur spontaneously A system is at equilibrium and no net change can occur if G is zero A reaction will not occur spontaneously if G is positive; to proceed, it must receive an input of free energy from another source. 15 G is the Gibbs free energy change, used to describe energetics of biochemical reactions H is the change in enthalpy, heat content of a system. S is the change in entropy, a measure of the level of randomness or disorder in a system. The total entropy of a system and its surroundings always increases for a spontaneous process) {2 nd Law} Entropy will increase only if: G = H system - T S system < 0 The free-energy change must be negative for a reaction to be spontaneous G = H system - T S system 16 G of a reaction depends only on free-energy of products minus free-energy of reactants. G of a reaction is independent of path (or molecular mechanism) of the transformation G provides no information about the rate of a reaction For the reaction: A + B C + D To determine G, must consider nature of both reactants and products as well as their concentrations 17 F REE ENERGY & EQUILIBRIUM CONSTANT (K) At equilibrium, G = 0. 0 = G o + RTln([C][D]/[A][B]) K eq = [C][D]/[A][B] G o = - RTlnK eq An enzyme cannot alter the equilibrium of a chemical reaction. This means, an enzyme accelerates the forward and reverse reactions by precisely the same factor. 18 19 20 21 E NZYMES DECREASE ACTIVATION ENERGY A chemical reaction goes through a transition state with a higher G than either S of P Enzymes facilitate the formation of the transition state by decreasing G 22 E NZYME -S UBSTRATE C OMPLEX For enzymes to function, they must come in contact with the substrate. While in contact, they are referred to as the enzyme-substrate complex. The combination of substrate and enzyme creates a new reaction pathway, with a lowered transition-state energy More molecules have the required energy to reach the transition state Catalytic power of enzymes is derived from the formation of the transition states in enzyme-substrate (ES) complexes The essence of catalysis is specific binding of the transition state 23 E NZYME A CTIVE S ITE Enzymes are often quite large compared to their substrates. The relatively small region where the substrate binds and catalysis takes place is called the active site. (e.g., human carbonic anhydrase:) 24 E NZYME A CTIVE S ITE The active site is the region that binds the substrates (& cofactors if any) It contains the residues that directly participate in the making & breaking of bonds (these residues are called catalytic groups) The interaction of the enzyme and substrate at the active site promotes the formation of the transition state The active site is the region that most directly lowers G of the reaction - resulting in rate enhancement of the reaction 25 26 E NZYMES DIFFER WIDELY IN, STRUCTURE, SPECIFICITY, & MODE OF CATALYSIS, YET, ACTIVE SITE HAVE COMMON FEATURES : The active site is a 3-D cleft formed by groups that come from different parts of the amino acid sequence Water is usually excluded unless it is a reactant. Substrates bind to enzymes by multiple weak attractions (electrostatic interactions, hydrogen bonds, hydrophobic interactions, etc. The specificity of binding depends on the precisely defined arrangement of atoms at the active site E NZYME C LASSIFICATION Enzymes are classified and named according to the types of reactions they catalyze: Proteolytic enzymes [such as trypsin] lyse protein peptide bonds. ATPase breaks down ATP ATP synthetase synthesizes ATP Lactate dehydrogenase oxidizes lactate, removing two hydrogen atoms. Such a wide variety of names can be confusing. A better method was needed. 27 E NZYME C LASSIFICATION The Enzyme Commission invented a systematic numbering system for enzymes based upon these categories, with extensions for various subgroups. e.g., nucleoside monophosphate kinase (transfers phosphates) EC = Transferase, 7 = phosphate transferred, 4=transferred to another phosphate, 4 = acceptor 28 29 LIVING ORGANISMS NEED ENERGY FOR: 1.Performing mechanical work 2.Active transport and maintaining homeostasis 3.Synthesis of macromolecuels and biochemicals. 30 METABOLIC PATHWAYS 1.Catabolic pathways 2.Anabolic pathways 3.Amphibolic pathways that can be both anabolic and catabolic depending on the energy status in the cell. 31 T HE USEFUL FORMS OF ENERGY PRODUCED IN CATABOLISM ARE EMPLOYED IN ANABOLISM 32 A METABOLIC PATHWAY MUST SATISFY MINIMALLY TWO CRITERIA 1.The reactions of the pathway must be specific: This criterion is accomplished by enzymes 2.The entire set of reactions must be thermodynamically favored. A reaction can occur spontaneously only if G, the change in free energy, is negative. 33 AN IMPORTANT THERMODYNAMIC FACT The overall free-energy change for a chemically coupled series of reactions is equal to the sum of the free energy changes of the individual steps. 34 The two reactions are catalyzed by the same enzyme 35 36 37 38 39 ATP IS THE ENERGY CURRENCY IN BIOLOGICAL SYSTEMS 40 The active form of ATP is usually attached to Mg 2+ or Mn 2+ 41 ATP IS THE ENERGY CURRENCY IN BIOLOGICAL SYSTEMS ATP is an energy-rich molecule because its triphosphate unit contains two phosphoanhydride bonds. A large amount of free energy is liberated when ATP is hydrolyzed to ADP and (Pi) or to AMP and PPi. 42 ATP IS THE ENERGY CURRENCY IN BIOLOGICAL SYSTEMS The precise G for these reactions depends on: The ionic strength of the medium The concentrations of Mg2+ and other metal ions. Under typical cellular concentrations, the actual G for these hydrolyses is approximately -12 kcal mol-1 (-50 kJ/mol). ATP hydrolysis can be coupled to promote unfavorable reactions. 43 ATP H YDROLYSIS D RIVES M ETABOLISM BY S HIFTING THE E QUILIBRIUM OF C OUPLED R EACTIONS 44 A CAN BE CONVERTED INTO B IF THE REACTION IS COUPLED TO ATP HYDROLYSIS 45 ATP IS AN ENERGY COUPLING AGENT : The hydrolysis of an ATP molecule in a coupled reaction changes the equilibrium by a factor of 10 8 (1.15 X10 -3 compared to 1.34X10 5 ) 46 A and B in the preceding example can be any two different chemical species A and B may represent activated and unactivated conformations of a protein phosphorylation with ATP may be a means of conversion into an activated conformation. Such a conformation can store free energy, which can then be used to drive a thermodynamically unfavorable reaction. (muscle contraction). A and B may refer to the concentrations of an ion or molecule on the outside and inside of a cell, as in the active transport of a nutrient. 47 T HE S TRUCTURAL B ASIS O F T HE H IGH P HOSPHORYL T RANSFER P OTENTIAL O F ATP 1.Resonance stabilization: ADP and, particularly, Pi, have greater resonance stabilization than does ATP. 2.Electrostatic repulsion: At pH 7, the triphosphate unit of ATP carries about four negative charges in close proximity. The repulsion between them is reduced when ATP is hydrolyzed. 3.Stabilization due to hydration: Water can bind more effectively to ADP and Pi than it can to the phosphoanhydride part of ATP, stabilizing the ADP and Pi by hydration. P HOSPHORYL T RANSFER P OTENTIAL 48 49 P HOSPHORYL T RANSFER P OTENTIAL It is significant that ATP has an intermediate phosphoryl transfer potential among the biologically important phosphorylated molecules. This intermediate position enables ATP to function efficiently as a carrier of phosphoryl groups. If ATP had the highest potential then it wouldnt be formed. 50 S OURCES OF ATP DURING EXERCISE 51 In resting muscle, [ATP] = 4 mM, [creatine phosphate] = 25 mM [ATP] sufficient to sustain 1second of muscle contraction ATP-ADP CYCLE 52 Only 100g of ATP in the body, turnover is very high. This amount must be constantly recycled every day. The ultimate source of energy for constructing ATP is food; ATP is simply the carrier and regulation-storage unit of energy. The average daily intake of 2,500 food calories translates into a turnover of a 180 kg of ATP Resting human consumes 40 kg of ATP in 24 hours. Strenuous exertion: 0.5 kg / minute. 2hr run: 60kg utilized C ARBON F UELS - A N IMPORTANT S OURCE OF C ELLULAR E NERGY The more reduced a carbon is, the more energy its oxidation will give. 53 Fats are a more efficient fuel source than carbohydrates such as glucose due to the fact that carbon in fats is more reduced. Energy derived from carbon oxidation is used in: creating a high phosphoryl transfer potential compound creating an ion gradient. In both cases, the end point is the formation of ATP. 54 T HE COMPLEXITY OF METABOLISM IS SIMPLIFIED BY UNIFYING THEMES The use of activated carriers is a recurring motif in biochemistry: - Weve seen that phosphoryl transfer can be used to drive otherwise endergonic reactions, - alter the energy of conformation of a protein, - or serve as a signal to alter the activity of a protein. The phosphoryl-group donor in all of these reactions is ATP. So ATP is an activated carrier of phosphoryl groups because it is an exergonic process.ATP 55 56 1.A CTIVATED ELECTRONS CARRIERS FOR FUEL OXIDATION. The reactive part of NAD+ is its nicotinamide ring, a pyridine derivative synthesized from the vitamin niacin. Nicotinamide adenine dinucleotide (NAD + ): R = H Nicotinamide adenine dinucleotide phosphate (NADP + ): R = PO 3 2- Nicotinamide Adenine Dinucleotide NAD + /NADH Oxidized forms 57 In the oxidation of a substrate, the nicotinamide ring of NAD + accepts a hydrogen ion and two electrons, which are equivalent to a hydride ion and becomes reduced. 58 Flavin Adenine Dinucleotide FAD/FADH 2 This electron carrier consists of a flavin mononucleotide (FMN) unit (shown in blue) and an AMP unit (shown in black). 59 FAD, like NAD +, can accept two electrons. In doing so, FAD, unlike NAD +, takes up two protons. 60 2.A N ACTIVATED CARRIER OF ELECTRONS FOR REDUCTIVE BIOSYNTHESIS NADPH carries electrons in the same way as NADH. NADPH is used almost exclusively for reductive biosyntheses, whereas NADH is used primarily for the generation of ATP. The extra phosphoryl group on NADPH is a tag that enables enzymes to distinguish between high-potential electrons to be used in anabolism and those to be used in catabolism. 61 3.A N ACTIVATED CARRIER OF TWO - CARBON FRAGMENTS. Coenzyme A, another central molecule in metabolism, is a carrier of acyl groups The terminal sulfhydryl group in CoA is the reactive site. Acyl groups are linked to CoA by thioester bonds. The resulting derivative is called an acyl CoA. 62 An acyl group often linked to CoA is the acetyl unit: The G for the hydrolysis of acetyl CoA has a large negative value: Acetyl CoA has a high acetyl group- transfer potential because transfer of the acetyl group is exergonic. A CTIVATED CARRIERS 63 A small set of carriers responsible for most interchanges of activated groups in metabolism 64 NADH, NADPH, and FADH 2 react slowly with O 2 in the absence of a catalyst. Likewise, ATP and acetyl CoA are hydrolyzed slowly in the absence of a catalyst. These molecules are kinetically quite stable in the face of a large thermodynamic driving force for reaction with O 2 (in regard to the electron carriers) and H 2 O (in regard to ATP and acetyl CoA). The kinetic stability of these molecules in the absence of specific catalysts enables enzymes to control the flow of free energy and reducing power. 65 K EY R EACTIONS A RE R EOCCURRING T HROUGHOUT M ETABOLISM Types of chemical reactions in metabolism: 66 1.O XIDATION - REDUCTION REACTIONS Reduction: Gain of electrons Gain of hydrogen Loss of oxygen Oxidation: Loss of electrons Loss of hydrogen Gain of oxygen 67 2.L IGATION REACTIONS Bond formation using free energy from ATP cleavage. 3.I SOMERIZATION REACTIONS Rearrange particular atoms within the molecule. Their role is often to prepare a molecule for subsequent reactions such as the oxidation- reduction. 68 4.G ROUP - TRANSFER REACTIONS 69 5.H YDROLYTIC REACTIONS Cleave bonds by the addition of water. Hydrolysis is a common means employed to break down large molecules. 70 6.T HE A DDITION O F F UNCTIONAL G ROUPS T O D OUBLE B ONDS O R T HE R EMOVAL O F G ROUPS F ORM D OUBLE B ONDS 71 72 I MPORTANT GENERAL PRINCIPLE OF METABOLISM Biosynthetic and degradative pathways are almost always distinct. It facilitates the control of metabolism. Metabolic regulation and flexibility are enhanced by compartmentalization: For example, fatty acid oxidation takes place in mitochondria, whereas fatty acid synthesis takes place in the cytosol 73 M ETABOLIC P ROCESSES A RE R EGULATED IN T HREE P RINCIPAL W AYS 1.The amounts of enzymes 2.Their catalytic activities 3.The accessibility of substrates 74 75 1.T HE AMOUNTS OF ENZYMES Rate of enzyme synthesis: Transcriptional regulation (ex. Lactose upregulates -Gal within minutes) Translational regulation Rate of enzyme degradation: RNA degradation Protein degradation 2.T HEIR CATALYTIC ACTIVITIES : Reversible allosteric control: Feed back inhibition of the first step of most metabolic pathways by the pathway products. Reversible covalent modification: Phosphorylation-dephosphorylation. Hormones like insulin and epinephrine trigger the reversible modifications of key enzymes. 76 3.T HE ACCESSIBILITY OF SUBSTRATES Controlling the flux of substrate Transfer of substrate from one compartment of the cell to the other. 77