ADVANCED BIOCHEMISTRY Metabolism FALL 2014

1. ADVANCED BIOCHEMISTRY Metabolism FALL 2014 IUG, Fall 2014

2. METABOLISMBASIC CONCEPTS & DESIGN The quantitative study of cellular energy transductions and the chemical reactions underlying these transductions. Or summation of all chemical reactions occurring in vivo

3. Enzymes • Enzymesare biological catalysts. • 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).

4. Enzyme Characteristics:Rate Enhancement • Enzymes significantly enhance the rates of reactions:

5. Enzyme Characteristics:Turnover Numbers • The “turnover number” is used to rate the effeciency of an enzyme. • It tells how many molecules of reactant per a molecule of enzyme can be converted to product(s) per second. • How fast can an enzyme produce products?

6. Enzyme Characteristics:Specificity • Enzymes can be very specific. • For example, proteolytic enzymes help hydrolyze peptide bonds in proteins. • Trypsin is very specific • Thrombin is too specific 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.

7. Enzyme Characteristics:Regulation • Enzymes can enhance the rates of reactions by many orders of magnitude. • A rate enhancement of 1017 means that what would occur in 1 second with an enzyme’s 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

8. Enzyme Cofactors • 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 “Apoenzyme” + cofactor = “Holoenzyme”

9. Enzymes – Gibbs Free Energy • Gibb’s “Free Energy,” ΔG, determines the spontaneity of a reaction: • ΔG must be negative for a reaction to occur spontaneously • A system is at equilibrium and no net change can occur if ΔG is zero • A reaction will not occur spontaneously if ΔG is positive; to proceed, it must receive an input of free energy from another source.

10. 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

11. Free energy & equilibrium constant (K) • At equilibrium, G = 0. • 0 = Go’ + RTln([C][D]/[A][B]) • K’eq =[C][D]/[A][B] • Go’ = - 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.

12. Enzymes 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‡

13. Enzyme-Substrate Complex • 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 (جوهر العملية التحفزية)is specific binding of the transition state

14. Enzyme – Active Site • 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:)

15. Enzyme – Active Site • 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

16. Enzymes 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

17. Enzyme Classification • Enzymes are classified and named according to the types of reactions they catalyze: • Proteolyticenzymes [such astrypsin] 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.

18. Enzyme Classification • 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 2.7.4.4. 2 = Transferase, 7 = phosphate transferred, 4=transferred to another phosphate, 4 = acceptor

19. LIVING ORGANISMS NEED ENERGY FOR: • Performing mechanical work • Active transport and maintaining homeostasis • Synthesis of macromolecuels and biochemicals.

20. METABOLIC PATHWAYS • Catabolic pathways • Anabolic pathways • Amphibolic pathways that can be both anabolic and catabolic depending on the energy status in the cell.

21. The useful forms of energy produced in catabolism are employed in anabolism

22. A METABOLIC PATHWAY MUST SATISFY MINIMALLY TWO CRITERIA • The reactions of the pathway must be specific: • This criterion is accomplished by enzymes • The entire set of reactions must be thermodynamically favored. • A reaction can occur spontaneously only if DG, the change in free energy, is negative.

23. 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.

24. ATP IS THE ENERGY CURRENCY IN BIOLOGICAL SYSTEMS The active form of ATP is usually attached to Mg2+ or Mn2+

25. 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.

26. ATP IS THE ENERGY CURRENCY IN BIOLOGICAL SYSTEMS • The precise DG°’ 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 DG for these hydrolyses is approximately -12 kcal mol-1 (-50 kJ/mol). • ATP hydrolysis can be coupled to promote unfavorable reactions.

27. ATP Hydrolysis Drives Metabolism by Shifting the Equilibrium of Coupled Reactions

28. A can be converted into B if the reaction is coupled to ATP hydrolysis

29. ATP is an energy coupling agent: The hydrolysis of an ATP molecule in a coupled reaction changes the equilibrium by a factor of 108 (1.15 X10-3 compared to 1.34X105)

30. 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.

31. The Structural Basis Of The High Phosphoryl Transfer Potential Of ATP • Resonance stabilization: • ADP and, particularly, Pi, have greater resonance stabilization than does ATP. • 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. • 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.

32. Phosphoryl Transfer Potential

33. Phosphoryl Transfer Potential • 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 wouldn’t be formed.

35. Sources of ATP during exercise In resting muscle, [ATP] = 4 mM, [creatine phosphate] = 25 mM [ATP] sufficient to sustain 1second of muscle contraction

36. ATP-ADP cycle • 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

37. Carbon Fuels- An important Source of Cellular Energy • The more reduced a carbon is, the more energy its oxidation will give.

38. 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.

39. The complexity of metabolism is simplified by unifying themes • The use of activated carriers is a recurring motif in biochemistry: - We’ve 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.

40. Activated electrons carriers for fuel oxidation. Nicotinamide Adenine Dinucleotide NAD+/NADH 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 = PO32- Oxidized forms

41. 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.

42. Flavin Adenine Dinucleotide FAD/FADH2 This electron carrier consists of a flavin mononucleotide (FMN) unit (shown in blue) and an AMP unit (shown in black).

43. FAD, like NAD+, can accept two electrons. In doing so, FAD, unlike NAD+, takes up two protons.

44. An 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.

45. An 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.

46. An acyl group often linked to CoA is the acetyl unit: • The DG° 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.

47. Activated carriers A small set of carriers responsible for most interchanges of activated groups in metabolism

48. NADH, NADPH, and FADH2 react slowly with O2 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 O2 (in regard to the electron carriers) and H2O (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.

49. Key Reactions Are Reoccurring Throughout Metabolism Types of chemical reactions in metabolism:

50. Oxidation-reduction reactions • Reduction: • Gain of electrons • Gain of hydrogen • Loss of oxygen • Oxidation: • Loss of electrons • Loss of hydrogen • Gain of oxygen Reduced Oxidized