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BIOC 299 Final: UVic Biochem 299 Final exam NOTES

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BIOC 299
Briant Douglas

Chapter 18: Preparation for the Cycle • 18.1 Pyruvate Dehydrogenase Forms Acetyl Coenzyme A from Pyruvate Most fuel molecules enter the citric acid cycle as acetyl CoA. The link between glycolysis and the citric acid cycle is the oxidative decarboxylation of pyruvate to form acetyl CoA. In eukaryotes, this reaction takes place inside mitochondria, in contrast with glycolysis, which takes place in the cytoplasm. The enzyme complex catalyzing this reaction, the pyruvate dehydrogenase complex, consists of three distinct enzyme activities. Pyruvate dehydrogenase catalyzes the decarboxylation of pyruvate and the formation of acetyllipoamide. Dihydrolipoyl transacetylase forms acetyl CoA, and dihydrolipoyl dehydrogenase regenerates the active transacetylase. The complex requires five cofactors: thiamine pyrophosphate, lipoic acid, coenzyme A, NAD+, and FAD. • 18.2 The Pyruvate Dehydrogenase Complex is Regulated by Two Mechanisms The irreversible formation of acetyl CoA from pyruvate is an important regulatory point for the entry of glucose-derived pyruvate into the citric acid cycle. The pyruvate dehydrogenase complex is regulated by feedback inhibition by acetyl CoA and NADH. The activity of the pyruvate dehydrogenase complex is stringently controlled by reversible phosphorylation by an associated kinase and phosphatase. High concentrations of ATP and NADH stimulate the kinase, which phosphorylates and inactivated the complex. The importance of the pyruvate dehydrogenase complex to metabolism, especially to catabolism in the central nervous system, is illustrated by beriberi. Beriberi is a neurological condition that results from a deficiency of thiamine, the vitamin precursor of thiamine pyrophosphate. The lack of TPP impairs the activity of pyruvate dehydrogenase component of the pyruvate dehydrogenase complex. Arsenite and mercury are toxic because of their effects on the complex. These chemicals bind to the lipoic acid coenzyme of the dihydrolipoyl dehydrogenase, inhibiting the activity of this enzyme. Chapter 19: Harvesting Electrons from the Cycle • 19.1 The Citric Acid Cycle Consists of Two Stages The first stage of the citric acid cycle consists of the condensation of acetyl CoA with oxaloacetate, followed by two oxidative decarboxylations. In the second stage of the cycle, oxaloacetate is regenerated, coupled with the formation of high-transfer-potential electrons and a molecule of ATP or GTP. • 19.2 Stage One Oxidizes Two carbon Atoms to Gather Energy-Rich Electrons The cycle starts with the condensation of oxaloacetate (containing four carbon atoms, abbreviated as C 4 and acetyl CoA (C ) 2o give citrate (C )6 which is isomerized to isocitrate (C 6. Oxidative decarboxylation of this intermediate gives α-ketogluterate (C ).5The second molecule of carbon dioxide comes off in the next reaction, in which α-ketogluterate is oxidatively decarboxylated to succinyl CoA (C ).4 • 19.3 Stage Two Regenerated Oxaloacetate and Harvests Energy-Rich Electrons The Thioester bond of succinyl CoA is cleaved by orthophosphate to yield succinate, and a high-phosphoryl-transfer-potential compound in the form of ATP or GTP is concomitantly generated. Succinate is oxidized to fumerate (C )4 which is then hydrated to form malate (C ).4 Finally, malate is oxidized to regenerate oxaloacetate (C 4. Thus, two carbon atoms from acetyl CoA enter the cycle, and two carbon atoms leave the cycle as CO in t2e successive decarboxylations catalyzed by isocitrate dehydrogenase and α-ketogluterate dehydrogenase. In the four oxidation-reduction reactions in the cycle, three pairs of electrons are transferred to NAD and one pair to FAD. These reduced electron carriers are subsequently oxidized by the electron-transport chain to generate approximately 9 molecules of ATP. • 19.4 The Citric Acid Cycle Is Regulated The citric acid cycle operates only under aerobic conditions because it requires a supply of NAD and FAD. The irreversible formation of acetyl CoA from pyruvate is an important regulatory point for the entry of glucose-derived pyruvate into the citric acid cycle. The electron acceptors are regenerated when NADH and FADH transfer2their electrons to O through 2he electron-transport chain, with the concomitant production of ATP. Consequently, the rate of the citric acid cycle depends on the need for ATP. In eukaryotes, the regulation of two enzymes in the cycle also is important for control. A high-energy charge diminishes the activities of isocitrate dehydrogenase and α-ketogluterate dehydrogenase. These mechanisms complement each other in reducing the rate of formation of acetyl CoA when the energy charge of the cell is high and when the biosynthetic intermediates are abundant. • 19.5 The Glyoxylate Cycle Enables Plants and Bacteria to Convert Fats into Carbohydrates The glyoxylate cycle enhances the metabolic versatility of many plants and bacteria. This cycle, which uses some of the reaction of the citric acid cycle, enables these organisms to convert fat into glucose. Two molecules of acetyl CoA are converted into succinate, which can be used to synthesize glucose. Chapter 20: The Electron-Transport Chain • 20.1 Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria Mitochondria generate most of the ATP required by aerobic cells through a joint endeavor of the reaction of the citric acid cycle, which takes place in the mitochondrial matrix, and oxidative phosphorylation, which takes place in the inner mitochondrial membrane. • 20.2 Oxidative Phosphorylation Depends on Electron Transfer In oxidative phosphorylation, the synthesis of ATP is coupled to the flow of electrons from NADH or FADH to O b2 a pr2ton gradient across the inner mitochondrial membrane. Electron flow through three asymmetrically oriented transmembrane complexes results in the pumping of protons out of the mitochondrial matrix and the generation of a membrane potential. ATP is synthesized when protons flow back to the matrix. • 20.3 The Respiratory Chain consists of Proton Pumps and a physical Link to the Citric Acid Cycle The electron carriers in the respiratory assembly of the inner mitochondrial membrane are quinones, flavins, iron-sulfur complexes, heme groups of cytochromes, and copper ions. Electrons from NADH are transferred to the FMN prosthetic group of NADH-! Oxioreductase (Complex I), the first of four complexes. This oxioreductase also contains Fe-S centers. The electrons emerge in QH , 2he reduced form of ubiquinone (Q). The citric acid cycle enzyme succinate dehydrogenase is a component of the succinate-Q reductase complex (Complex II), which donates electrons from FADH to Q2to form QH . This 2ighly mobile hydrophobic carrier transfers its electrons to Q-cytochrome c oxioreductase (Complex III), a complex that contains cytochromes b and c a1d an Fe-S center. This complex reduces cytochrome c, a water-soluble peripheral membrane protein. Cytochrome c, like Q, is a mobile carrier of electrons, which it then transfers to cytochrome c oxidase (Complex IV). This complex contains cytochromes a and a3and three copper ions. A heme iron ion and a copper ion in this oxidase transfer electrons to O 2 the ultimate acceptor, to form H 2. Chapter 21: The Proton-Motive Force • 21.1 A Proton Gradient Powers the Synthesis of ATP The flow of electrons through Complexes I, III, and IV of the electron transport chain leads to the transfer of protons from the matrix side to the cytoplasmic side of the inner mitochondrial membrane. A proton motive force consisting of a pH gradient (matrix side basic) and a membrane potential (matrix side negative) is generated. The flow of protons back to the matrix side through ATP synthase drives ATP synthesis. The enzyme complex is a molecular motor made of two operational units: a rotating component and a stationary component. The rotation of the γ subunit induced structural changes in the β subunit that result in the synthesis and release of ATP from the enzyme. Proton influx provides the force for the rotation. The flow of two electrons through NADH-Q oxioreductase, Q-cytochrome c oxioreductase, and cytochrome c oxidase regenerated a gradient sufficient to synthesize 1, 1, and 0.5 molecule of NADH oxidized in the mitochondrial matrix, whereas only 1.5 molecules of ATP are made per molecule of FADH oxidi2ed, because its electrons enter the chain at QH , 2 after the first proton-pumping site. • 21.2 Shuttles Allow Movement Across Mitochondrial Membranes Mitochondria employ a host of transporters, or carriers, to move molecules across the inner mitochondrial membrane. The electrons of cytoplasmic NADH are transferred into the mitochondria by the glycerol 3-phosphate shuttle to form FADH from2FAD or by the malate- aspartate shuttle to form mitochondrial NADH. The entry of ADP into the mitochondrial matrix is coupled to the exit of ATP by ATP-ADP translocase, a transporter driven by membrane potential. • 21.3 Cellular Respiration is Regulated by the Need for ATP About 30 molecules of ATP are generated when a molecule of glucose is completely oxidized to CO 2nd H O.2Electron transport is normally tightly coupled to phosphorylation. NADH and FADH are o2idized only if ATP is simultaneously phosphorylated to ATP, a form of regulation called acceptor or respiratory control. Proteins have been identified that uncouple electron transport and ATP synthesis for the generation of heat. Uncouplers such as 2,4- dinitrophenol also can disrupt this coupling; they dissipate the proton gradient by carrying protons across the inner mitochondrial membrane. Chapter 33: The Structure of Informational Macromolecules: DNA and RNA • 33.1 A Nucleic Acid Consists of Bases Linked to a Sugar-Phosphate Backbone DNA and RNA are linear polymers of a limited number of monomers. In DNA, the repeating units are nucleotides with the sugar being a deoxyribose and the bases being adenine, thymine, guanine, and cytosine. In RNA, the sugar is a ribose and the base uracil is used in place of thymine. DNA is the molecule of heredity in all prokaryotic and eukaryotic organisms. • 33.2 Nucleic Acid Strands Can Form a double-Helical Structure All cellular DNA consists of two very long, helical polynucleotide strands coiled around a common axis. The sugar-phosphate backbone of each strand is on the outside of the double helix, whereas the purine and pyrimidine bases are on the inside. The two strands of the double helix run in opposite directions. The two strands are held together by hydrogen bonds between pairs of bases: adenine is always paired with thymine, and guanine is always paired with cytosine. The sequence of one strand thus determines the sequence of the other. This sequence determination makes DNA an especially appropriate molecule for storing genetic information, which is the precise sequence of bases along a strand. The strands can be separated, and each separated strand can be used to make a double helix identical with the original molecule. • 33.3 DNA Double Helices Can Adopt Multiple Forms DNA is a structurally dynamic molecule that can exist in a variety of helical forms: A- DNA, B-DNA (the classic Watson-Crick helix), and Z-DNA. DNA can be bent, kinked, and unwound. In A-, B-, and Z-DNA, two antiparallel strands are held together by Watson-Crick base pairs and by stacking interactions between bases in the same strand. The sugar-phosphate backbone is on the outside of the helix, and the bases are inside. A- and B-DNA are right-handed helices. In B-DNA, the base pairs are nearly perpendicular to the helix axis. An important structural feature of the B helix is the presence of major and minor grooves, which display different potential hydrogen-bond acceptors, and donors according to the base sequence. Most of the DNA in a cell is in the B-form. Double-stranded DNA can also wrap around itself to form supercoiled structures. The supercoiling of DNA has two important consequences. Supercoiling compacts the DNA and, because supercoiling is left-handed, the DNA is partly unwound and more accessible for process such as replication and transcription. • 33.4 Eukaryotic DNA is Associated with Specific Proteins Eukaryotic DNA is tightly bound to proteins, most notably to a group of small basic proteins called histones. The entire complex of a cell’s DNA and associated protein is called chromatin. Five major histones are present in chromatin: four histones-H2A, H2B, H3, and H4- associate with one another; the other histone is H1. Chromatin is made up of nucleosomes- repeating units; each containing 200bp of DNA and two copies each of H2A, H2B, H3, and H4. The DNA forms a left-handed super helix as it wraps around the outside of a nucleosome. The winding of DNA around the nucleosome core contributes to DNA’s packing. The nucleosomes themselves are arranged in a helical array. • 33.5 RNA Can adopt Elaborate Structures Although RNA is usually single stranded, the single strands fold back on itself to form elaborate structures. The simplest structure is the stem-loop stabilized by Watson-Crick base pairs. In other cases, hydrogen-bond donors and acceptors that are not normal participants in Watson-Crick base pairs form hydrogen bonds in nonstandard pairings. Chapter 34: DNA Replication • 34.1 DNA is Replicated by Polymerases DNA polymerases are template-directed enzymes that catalyze the formation of phosphodiester linkages by the 3’-hydroxyl group’s nucleophilic attack on the innermost phosphorus atom of a deoxyribonucleoside 5’-triphosphate. They cannot start strands de novo; a primer with a free 3’-hydroxyl group is required. Many DNA polymerases proofread that nascent product: their 3’-5’ exonuclease activity potentially edits the outcome of each polymerization step. A mispaired nucleotide is excised before the next step proceeds. In E. coli, DNA polymerase I repairs DNA and participates in replication. Fidelity is further enhanced by an induced fit that results in a catalytically active conformation only when the complex of enzyme, DNA, and correct dNTP is formed. • 34.2 DNA Replication is Highly Coordinated DNA replication is E. coli starts at a unique origin (oriC) and proceeds sequentially in opposite directions. More than 20 proteins are required for replication. An ATP-driven helicase unwinds the oriC region to create a replication fork. At this fork, both strands of parental DNA serve as templates for the synthesis of new DNA. A short stretch of RNA formed by the primase, an RNA polymerase, primes DNA synthesis. One strand of DNA (the leading strand) is synthesized continuously, whereas the other strand (the lagging strand) is synthesized discontinuously, in the form of 1-kb Okazaki fragments. Both new strands are formed simultaneously by the concerted actions of the highly processive DNA polymerase III holoenzyme, an asymmetric dimer. The discontinuous assembly of the lagging strand enables 5’- 3’ polymerization at the molecular level to give rise to overall growth of this strand in the 3’-5’ direction. The RNA primer stretch is hydrolyzed by the 5’-3’ nuclease activity of DNA polymerase I, which also fills gaps. Finally, nascent DNA fragments are joined by DNA ligase in a reaction driven by ATP. DNA synthesis in eukaryotes is more complex than in bacteria. Eukaryotes require thousands of origins of replication to compete replication in a timely fashion. A special RNA- dependent DNA polymerase called telomerase is responsible for the replication of the ends of linear chromosomes. Chapter 35: DNA Repair and Recombination • 35.1 Errors Can Arise in DNA Replication DNA can be damage in a variety of different ways. For example, mismatched bases may be incorporated in the course of DNA replication or individual bases may be damaged by oxidation or by hydrocarbon attachment after DNA replication. Other forms of damage are the formation of cross-links and the introduction of single- or double-stranded breaks in the DNA backbone. • 35.2 DNA Damage can be Detected and Repaired Several different repair systems detect and repair DNA damage. Repair begins with the process of proofreading in DNA replication: mismatched bases that were incorporated in the course of synthesis are excised by exonuclease activity present in replicative polymerases. Some DNA lesions such as thymine dimers can be directly reversed through the action of specific enzymes. Other DNA-repair pathways act through the excision of single damaged bases (base- excision repair) or short segments of nucleotides (nucleotide-excision repair). Defects in DNA- repair components are associated with susceptibility to many different sorts of cancer. Many potential carcinogens can be detected by their mutagenic action on bacteria (the Ames test). • 35.3 DNA Recombination Plays Important Roles in Replication and Repair Recombination is the exchange of segments between two DNA molecules. It is important in some types of DNA repair as well as other processes such as meiosis, the generation of antibody diversity, and the lifecycles of some viruses. Some recombination pathways are initiated by strand invasion, in which a single strand at the end of a DNA double helix forms base pairs with one stand in another double helix and displaces the other strand. A common intermediate formed in other recombination pathways is the Holliday junction, which consists of four strands of DNA that come together to form a crosslike structure. Chapter 36: RNA Synthesis and Regulation in Bacteria • 36.1 Cellular RNA is Synthesized in RNA Polymerases All cellular RNA molecules are synthesized by RNA polymerases according to instructions given by DNA templates. The activated monomer substrates are ribonucleoside triphosphates. The direction of RNA synthesis is 5’3’, as in DNA synthesis. RNA polymerases, unlike DNA polymerases, do not need a primer. RNA polymerase in E. coli is a multisubstituent enzyme. The subunit composition of the ~500-kd holoenzyme is 𝜶 𝜷𝜷′𝝈𝟐 and that of the core enzyme is 𝜶𝟐𝜷𝜷′𝝎. • 36.2 RNA Synthesis Comprises Three Stages Transcription is initiated at promoter sites consisting of two sequences, one centered near -10 and the other near -35; that is, 10 and 35 nucleotides away from the start site in the 5’ (upstream) direction, respectively. The consensus sequence of the -10 regions is TATAAT. The
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