15-Biochemistry-Lecture15.pdf

METABOLISM IS THE SUM OF ALL CHEMICAL REACTIONS IN THE CELL Series of related reactions form metabolic pathways Some pathways are primarily breaking down complex structures for energy production – This is catabolism or breakdown Some pathways are primarily using energy to build complex structures –...

METABOLISM IS THE SUM OF ALL CHEMICAL REACTIONS IN THE CELL Series of related reactions form metabolic pathways Some pathways are primarily breaking down complex structures for energy production – This is catabolism or breakdown Some pathways are primarily using energy to build complex structures – This is anabolism or biosynthesis METABOLISM IS THE SUM OF ALL CHEMICAL REACTIONS IN THE CELL Energy relationships between catabolic and anabolic pathways § Catabolism: Degradation of nutrients macromolecules (carbohydrates, fats and proteins) provides sources of energy and metabolic precursors for anabolism. § Anabolism: Synthesis of cell macromolecules from precursors (e.g., essential amino acids for proteins, precursors for lipids, etc.). THREE TYPES OF NONLINEAR METABOLIC PATHWAYS Converging (catabolic), diverging (anabolic), and cyclic pathways Oxaloacetate (one of the starting materials) is regenerated and re-enters the pathway (c). Acetate, a key metabolic intermediate is the breakdown product of many fuels (a) Serves as the precursor for an array of products (b) Is consumed in the catabolic pathway of the Citric Acid Cycle. MAP OF METABOLIC PATHWAYS Metabolism as a three-dimensional meshwork. A typical eukaryotic cell has the capacity to make about 30,000 different proteins, which catalyze thousands of different reactions involving many hundreds of metabolites, most shared by more than one “pathway. In this much-simplified overview of metabolic pathways, each dot represents an intermediate compound and each connecting line represents an enzymatic reaction. For a more realistic and far more complex diagram of metabolism, see the online interactive map KEGG PATHWAY database:https://www.genome.jp/pathway/ map01100 LAWS OF THERMODYNAMICS APPLY TO LIVING ORGANISMS Living organisms cannot create energy from nothing Living organisms cannot destroy energy into nothing Living organism may transform energy from one form to another – In the process of transforming energy, living organisms must increase the entropy of the universe In order to maintain organization within themselves, living systems must be able to extract useable energy from their surroundings, and release “useless” energy (heat) back to their surroundings STANDARD FREE ENERGY (DG’0), OR THE EQUILIBRIUM CONSTANT (K’eq), MEASURE THE DIRECTION OF PROCESSES DG’o = -RT ln K’eq STANDARD FREE ENERGY OF SOME CHEMICAL REACTIONS ENERGETICS OF SOME CHEMICAL REACTIONS Hydrolysis reactions tend to be strongly favorable (spontaneous): DG’° < 0 Isomerization reactions have smaller free-energy changes – Isomerization between enantiomers: DG’° = 0 Complete oxidation of reduced compounds is strongly favorable DG’° < 0 – In biochemistry the oxidation of reduced fuels with O2 is stepwise and controlled – Recall that being thermodynamically favorable is not the same as being kinetically rapid ENERGETICS WITHIN THE CELL ARE NOT STANDARD The actual free-energy change (DG) of a reaction in the cell depends on: – 1/ The standard change in free energy (DG’0) in standard conditions (25°C) and at equilibrium (Keq) – 2/ Actual concentrations of products and reactants – For example: Reaction aA + bB cC + dD: [C]c [D]d ΔG = ΔG '° + RT ln [A]a [B]b Standard free-energy changes are additive: for two sequential chemical reactions, A ⇄ B and B ⇄ C, each chemical reaction has its own equilibrium constant and standard free-energy change ∆G1′° and ∆G2′° for the overall reaction, A ⇄ C, the standard free-energy change is the sum of the individual standard free-energy changes GROUP TRANSFER REACTIONS IN BIOCHEMISTRY Proton transfer: very common Methyl transfer: various biosynthesis Acyl transfer: biosynthesis of fatty acids Glycosyl transfer: attachment of sugars Phosphoryl transfer: to activate metabolites ‒ also important in signal transduction PHOSPHORYL TRANSFER FROM ATP (Phosphoryl transfer reactions are required for the activation of molecules for reactions that would otherwise be highly unfavorable) In this substitution from phosphorous, nucleophile (R-OH) forms a partial bond to the phosphorous center giving a pentacovalent intermediate or a pentacoordinated transition state When a nucleophile Z (in this case, the —OH on C-6 of glucose) attacks ATP, it displaces ADP (W) , and becomes phosphorylated. HYDROLYSIS OF ATP IS HIGHLY FAVORABLE UNDER STANDARD CONDITIONS The free-energy change for ATP is large and negative Chemical basis for the large free-energy change associated with ATP hydrolysis 1/ Better charge separation: The charge separation that results from hydrolysis relieves electrostatic repulsion among the four negative charges on ATP. 2/ More favorable resonance stabilization of the products: The product inorganic phosphate (Pi) is stabilized by formation of a resonance hybrid, in which each of the four phosphorusoxygen bonds has the same degree of double-bond character and the hydrogen ion is not permanently associated with any one of the oxygens. (Some degree of resonance stabilization also occurs in phosphates involved in ester or anhydride linkages, but fewer resonance forms are possible than for Pi.) 3/ A third factor (not shown) that favors ATP hydrolysis is the greater degree of solvation (hydration) of the products Pi and ADP relative to ATP, which further stabilizes the products relative to the reactants. ACTUAL DG OF ATP HYDROLYSIS DIFFERS FROM DG°’ The actual free-energy change, DG, in a process depends on: – The standard free energy (DG°’) – The actual concentrations of reactants and products The free-energy change is more favorable if the reactant’s concentration exceeds its equilibrium concentration True reactant and the product are Mg-ATP and Mg-ADP, respectively [MgADP- ] × [Pi ] DG = DG °'+ RT ln [MgATP2- ] – DG’° also Mg++ dependent (see next slide) DG’° OF ATP HYDROLYSIS IS MG++ DEPENDENT Mg2+ and ATP: Formation of Mg2+ complexes partially shields the negative charges and influences the conformation of the phosphate groups in nucleotides such as ATP and ADP. ATP PROVIDES ENERGY BY GROUP TRANSFERS, NOT BY SIMPLE HYDROLYSIS ATP hydrolysis in two steps (a) The contribution of ATP to a reaction is often shown as a single step but is almost always a twosteps process. (a) Reaction catalyzed by ATPdependent glutamine synthetase: 1 A phosphoryl group is transferred from ATP to glutamate, then 2 the phosphoryl group is displaced by NH3 and released as Pi. ATP DONATES PHOSPHORYL, PYROPHOSPHORYL AND ADENYLYL GROUPS Nucleophilic displacement reactions of ATP: Any of the three P atoms (α, β, or γ) may serve as the electrophilic target for nucleophilic attack—in this case, by the labeled nucleophile R—18O: The nucleophile may be an alcohol (ROH), a carboxyl group (RCOO–), or a phosphoanhydride (a nucleoside mono- or diphosphate, for example). (a)When the oxygen of the nucleophile attacks the γ position, the bridge oxygen of the product is labeled, indicating that the group transferred from ATP is a phosphoryl (—PO32–), not a phosphate (—OPO32–). (b) Attack on the β position displaces AMP and leads to the transfer of a pyrophosphoryl (not pyrophosphate) group to the nucleophile. (c) Attack on the α position displaces PPi and transfers the adenylyl group to the nucleophile. CELLULAR CONCENTRATIONS OF ATP, ADP, AMP, AND Pi IN SOME CELLS Cellular ATP concentration depends on the type of cell, but it is usually far above the equilibrium concentration, making ATP a very potent source of chemical energy. OTHER PHOSPHORYLATED COMPOUNDS HAVE LARGE DG’° FOR HYDROLYSIS Hydrolysis of PhosphoEnolPyruvate (PEP) catalyzed by pyruvate kinase To ADP Large standard free energy of hydrolysis because: Electrostatic repulsion within the reactant molecule is relieved The products are stabilized via resonance (relative to the reactants), or by more favorable solvation The product undergoes further tautomerization Resonance stabilization of Pi also occurs. OTHER PHOSPHORYLATED COMPOUNDS HAVE LARGE DG’° FOR HYDROLYSIS Hydrolysis of 1,3-Bisphosphoglycerate (1,3-BPG) catalyzed by phosphoglycerate kinase To ADP Large standard free energy of hydrolysis because: – 3-phosphoglycerate has two resonance forms – removal of 3-phosphoglyceric acid by further metabolism and formation of the ion favor the forward reaction OTHER PHOSPHORYLATED COMPOUNDS HAVE LARGE DG’° FOR HYDROLYSIS Hydrolysis of Phosphocreatine (PCr) catalyzed by creatine kinase To ADP Large standard free energy of hydrolysis because: the release of Pi and the resonance stabilization of creatine favor the forward reaction BIOLOGICAL PHOSPHATES RANKING BY THE STANDARD FREE ENERGY OF HYDROLYSIS Phosphate can be transferred from compounds with higher ΔG’° to those with lower ΔG’°. Reactions such as: PEP + ADP => Pyruvate + ATP are favorable, and can be used to synthesize ATP. Flow of phosphoryl groups, represented by P, from highenergy phosphoryl group donors via ATP to acceptor molecules (such as glucose and glycerol) to form their low-energy phosphate derivatives. This flow of phosphoryl groups, catalyzed by kinases, proceeds with an overall loss of free energy under intracellular conditions. Hydrolysis of low-energy phosphate compounds releases Pi, which has an even lower phosphoryl group transfer potential. STANDARD FREE ENERGIES OF HYDROLYSIS OF SOME PHOSPHORYLATED COMPOUNDS AND ACETYL COA HYDROLYSIS OF THIOESTERS Hydrolysis of thioesters is strongly favorable – such as hydrolysis of acetyl-CoA Acetyl-CoA is an important donor of acyl groups – Feeding two-carbon units into metabolic pathways – Synthesis of fatty acids In acyl transfers, molecules other than water accept the acyl group HYDROLYSIS OF THIOESTERS Thioesters also have large free energies of hydrolysis Hydrolysis of acetyl-coenzyme A Acetyl-CoA is a thioester with a large, negative, standard free energy of hydrolysis. Thioesters contain a sulfur atom in the position occupied by an oxygen atom in oxygen esters. See the complete structure of coenzyme A elswhere. Comparison: Free energy of hydrolysis for thioesters and oxygen esters The products of both types of hydrolysis reaction have about the same free-energy content (G), but the thioester has a higher free-energy content than the oxygen ester. Orbital overlap between the O and C atoms allows resonance stabilization in oxygen esters; orbital overlap between S and C atoms is poorer and provides little resonnance stabilization: BIOLOGICAL OXYDATION-REDUCTION REACTIONS Loss of electrons by one chemical species (oxidized) corresponds to a gain of electrons by another (reduced) The flow of electrons in ox-red reactions is responsible for all work done by living organisms Oxidation states of carbon in the biosphere The oxidation states are illustrated with some representative compounds. Focus on the red carbon atom and its bonding electrons. When this carbon is bonded to the less electronegative H atom, both bonding electrons (red) are assigned to the carbon. When carbon is bonded to another carbon, bonding electrons are shared equally, so one of the two electrons is assigned to the red carbon. When the red carbon is bonded to the more electronegative O atom, the bonding electrons are assigned to the oxygen. The number to the right of each compound is the number of electrons "owned" by the red carbon, a rough expression of the oxidation state of that carbon. As the red carbon undergoes oxidation (loses electrons), the number gets smaller. BIOLOGICAL OXYDATION-REDUCTION REACTIONS Reduced organic compounds serve as fuels from which electrons can be stripped off during oxidation. The oxidation levels of carbon in biomolecules: Each compound is formed by oxidation of the red carbon in the compound shown immediately above. Carbon dioxide is the most highly oxidized form of carbon found in living systems. REVERSIBLE OXIDATION OF A SECONDARY ALCOHOL TO A KETONE Many biochemical oxidation-reduction reactions involve transfer of two electrons In order to keep charges in balance, proton transfer often accompanies electron transfer In many dehydrogenases, the reaction proceeds by a stepwise transfers of proton (H+) and hydride (:H–) An oxidation-reduction reaction. Shown here is the oxidation of lactate to pyruvate. In this dehydrogenation, two electrons and two hydrogen ions (the equivalent of two hydrogen atoms) are removed from C-2 of lactate, an alcohol, to form pyruvate, a ketone. In cells the reaction is catalyzed by lactate dehydrogenase and the electrons are transferred to the cofactor Nicotinamide Adenine Dinucleotide (NAD). One reactant gains electrons and is reduced while the other loses electrons and is oxidized This reaction is fully reversible; pyruvate can be reduced by electrons transferred from the cofactor. REDUCTION POTENTIALS OF SOME BIOLOGICAL REACTIONS Standard Reduction potential (E°’) measures affinity of a given compound for electrons (see Table for half reactions): – The higher the affinity, the higher the E°’ (more positive) – Electrons transferred from lower E°’ (donor) to higher (more positive) E°’ (acceptor) Difference in standard reduction potentials (for entire reactions): ∆E°’ – By convention ∆E°’ = E°’(e- acceptor) – E°’(e- donor) ∆E°’ can be used to calculate free energy change ∆G°’ : ∆G°’ = –nF∆E°’ ( DE°’= DG°’/nF = -(RT/nF)ln (Keq)) Difference in actual reduction potential (in cells): ∆E ∆E = ∆E°’ + RT/nF ln [products]/[reactants] (Nernst Eq.) Actual ∆G can be calculated from ∆E ∆G = –nF∆E. For negative DG, DE needs to be positive thus, E(acceptor) > E(donor) Or from ∆G°’ and actual concentrations of products and reactants in cells: ∆G = ∆G° + RT ln [products]/[reactants] NAD AND NADP, COMMON REDOX COFACTORS, ALLOW TWO LECTRONS TRANSFER These are commonly called pyridine nucleotide Cofactors and proteins serve as universal electron carriers: NADH and NADPH act with deshydrogenases as soluble, freely diffusible, electron carriers They can dissociate from the enzyme after the reaction In a typical biological oxidation reaction, hydride from an alcohol is transferred to NAD+ giving NAD-H Nicotinamide Adenine Dinucleotide, NAD+, and its phosphorylated analog NADP+ undergo reduction to NADH and NADPH, accepting a hydride ion (two electrons and one proton) from an oxidizable substrate. The hydride ion is added to either the front (the A side) or the back (the B side) of the planar nicotinamide ring. Note: The pyridine like rings of NAD and NADP are derived from the vitamin Niacin (Vit. B3), the deficiency of which causes Pellagra (3Ds: Dermatitis, Diarrhea and Dementia) FORMATION OF NADH CAN BE MONITORED BY UV-SPECTROPHOTOMETRY Measure the change of absorbance at 340 nm Very useful signal when studying the kinetics of NAD-dependent dehydrogenases The UV absorption spectra of NAD+ and NADH: Reduction of the nicotinamide ring produces a new, broad absorption band with a maximum at 340 nm. The production of NADH during an enzyme-catalyzed reaction can be conveniently followed by observing the appearance of the absorbance at 340 nm (molar extinction coefficient ε340 = 6,200 M–1cm–1). FLAVIN COFACTORS (FAD AND FMN) ALLOW SINGLE (OR DOUBLE) ELECTRON TRANSFERS Permits the use of molecular oxygen as an ultimate electron acceptor – flavin-dependent oxidases Flavin cofactors are tightly bound to proteins FAD and FMN can accept either one or two electrons and one or two protons. Oxidized and reduced FAD and FMN: FMN consists of the structure above the dashed line on the FAD (oxidized form). The flavin nucleotides accept two hydrogen atoms (two electrons and two protons), both of which appear in the flavin ring system. FAD and FMN can accept either one or two electrons and one or two protons. When FAD or FMN accepts only one hydrogen atom, the semiquinone, a stable free radical, forms. Note: The isoalloxazine ring of FAD and FMN are derived from the vitamin Riboflavin (Vit. B2) , the deficiency of which causes Stomatitis (similar to Pellagra). SUMMARY The rules of thermodynamics and organic chemistry still apply to living systems Reactions are favorable when the free energy of products is much lower than the free energy of reactants Biochemical phosphoryl transfer reactions are favorable when: – The phosphate donors are destabilized by electrostatic repulsion, – and the reaction products are often stabilized by resonance Unfavorable reactions can be made possible by chemically coupling a highly favorable reaction to the unfavorable reaction Oxidation-reduction reactions commonly involve transfer of electrons from reduced organic compounds to specialized redox cofactors – Reduced cofactors can be used in biosynthesis, or may serve as a source of energy for ATP synthesis