Biochemistry I 2014 Step 1 Review

1. Biochemistry I2014 Step 1 Review Monday, January 9th Seth Wander sawander@med.miami.edu

2. Biochemistry • Molecular Biology – nucleic acid + protein structure/processing • Cellular Biology – organelle structure/function, basic cellular processes • Nutrition/Metabolism – catabolic/anabolic pathways, metabolic disorders • Laboratory Techniques • Genetics – pedigree analysis, disease inheritance

3. Basic nucleic acid biology • Energy metabolism • Cell cycle • Genetics, pedigree analysis • Sample questions

4. Nucleic Acid Biology I • Purines (A,G) = 2 rings • Pyrimidines (C,T,U) = 1 ring • G-C *3 H-bonds • A-T *2 H-bonds • Cytosine deamination  uracil • *Base excision repair (why?) • Types of mutations: • Silent = same amino acid • Degenerate code, tRNA wobble • Missense = different amino acid (severity of mutation will vary) • Nonsense = Early stop codon (worst scenario, if truncation occurs early in sequence) • Frameshift = addition or deletion that is NOT a multiple of 3, alters reading frame.

5. Nucleic Acid Biology II • DNA replication, key enzymes: • Helicase – unwinds double helix @ origin (A-T rich) • Primase – establishes RNA primer (polymerase requires free 3’ end) • Topoisomerase – relieves supercoiling ahead of replication fork • DNA Polymerase III – elongates growing chain in 5’3’ direction, *includes 3’5’ proofreading for each new nucleotide • DNA Polymerase I – degrades RNA primers (5’3’ exonuclease) and replaces with DNA • Ligase – seals nicks in DNA between completed fragments • Telomerase – RNA-dependent DNA polymerase, extends chromosome ends

6. Nucleic Acid Biology III • Regulation of gene expression (DNA access): • Promoter – directly upstream from gene • TATA box, mutations alter transcript levels • Enhancers/Silencers – location varies dramatically due to DNA looping (may be VERY far, or even WITHIN gene) • Positive and negative transcription regulators will bind here • Chromatin architecture – histones (+) package DNA and actively regulate access of relevant enzymes • Heterochromatin – condensed, inactive • Euchromatin – open, active • DNA Repair (and clinical correlates): • Nucleotide Excision Repair – thymidine dimers, UV exposure  xeroderma pigmentosum • Base Excision Repair – cytosine deamination • Mismatch Repair – newly synthesized, **unmethylated strand is proofed for mismatched base pairs  hereditary nonpolyposis colon cancer

7. Nucleic Acid Biology IV • RNA processing in eukaryotes: • hnRNA  mRNA (occurs in nucleus) • 7-methylguanosine cap @ 5’ end • 3’ poly-AA tail (AAUAAA via poly-A polymerase) • Splicing - intron removal (spliceosome, snRNPs, lariat structure) • Protection from cytosolic anti-viral exonucleases • Regulation of mRNA longevity (eg. protein lifespan) • 20,000 – 40,000 genes  >150,000 proteins, how? • Alternative splicing, post-translational processing 5’ 5’ AAAAAAAA AAAAAAAA

8. Energy Metabolism I Cytoplasm Glucose Pentose Phosphate (HMP) Shunt Glucose-6-P Ribulose-5-P Glycolysis NADPH, Nulceotide sugars 2NAD+, 2ADP, 2Pi 2NADH, 2ATP Anaerobic 2x Pyruvate Lactate Fermentation Gluconeogenesis • NET: • Anaerobic glycolysis = 2ATP/ glucose • Aerobic Oxidative Phosphorylation = 34-36ATP/ glucose ATP Fatty Acids Acetyl-CoA e- Transport β Oxidation Fatty Acid Synthesis 3NADH, 1FADH2, 2CO2, 1GTP Aerobic TCA Mitochondrial Matrix

9. PyruvateDehydrogenase Complex I Pyruvate Acetyl-CoA • PDH deficiency • Congenital or acquired • **think alcoholic B1 deficiency • Lactic acidosis  neurological defects • Tx: ↑ ketogenic nutrients • (Lysine, Leucine) • ↑ dietary fat content • NET: bypass pyruvate, enter TCA directly NAD+, CoA NADH, CO2 • Key cofactors: • Pyrophosphate (B1, thiamine, TPP) • FAD (B2, riboflavin) • NAD (B3, niacin) • CoA (B5, pantothenate) • Lipoic Acid *** Contrast thiamine function with biotin Decarboxylation vs. carboxylation • PDH • α ketoglutarate dehydrogenase • branched chain ketoacid dehydrogenase • Pyruvate carboxylase • Acetyl-CoA carboxylase

10. PyruvateDehydrogenase Complex II Pyruvate Acetyl-CoA Thiamine (B1) deficiency (Alcoholics) = Wernicke-Korsakoff Syndrome NAD+, CoA NADH, CO2 • Wernicke • Lesion = foci of hemorrhage/necrosis @ mamillary bodies + periaqueductal grey matter • 1) Ophthalmoplegia (III, IV, VI) • 2) Ataxia • 3) Confusion • ** Reverse after thiamine admin! • Korsakoff • Lesion = deterioration @ dorsal medial nucleus (thalamus) • 1) Memory loss, anterograde amnesia • 2) Confabulation – fabricated stories to fill memory gaps, pts actually believe these events! • ** Typically permanent! Molecular biology: impaired glucose utilization via defective decarboxylation rxns.. PDH, α ketoglutarate DH, transketolase

11. Energy Metabolism I Cytoplasm Glucose Pentose Phosphate (HMP) Shunt Glucose-6-P Ribulose-5-P Glycolysis NADPH, Nulceotide sugars 2NAD+, 2ADP, 2Pi 2NADH, 2ATP Anaerobic 2x Pyruvate Lactate Fermentation Gluconeogenesis • NET: • Anaerobic glycolysis = 2ATP/ glucose • Aerobic Oxidative Phosphorylation = 34-36ATP/ glucose ATP Fatty Acids Acetyl-CoA e- Transport β Oxidation Fatty Acid Synthesis 3NADH, 1FADH2, 2CO2, 1GTP Aerobic TCA Mitochondrial Matrix

12. I II III IV Q C Electron Transport Chain 1 NADH 3 ATP, 1 FADH2 2 ATP H+ H+ Intermembrane Space H+ H+ H+ H+ ATP Synthase e- H+  H+ NADH NAD+ O2 H2O  FADH2 FADH H+ O2 ATP  ADP, Pi Mitochondrial matrix

13. I III IV Q C Electron Transport Chain 1 NADH 3 ATP, 1 FADH2 2 ATP H+ H+ Intermembrane Space H+ H+ H+ DNP H+ ATP Synthase Oligomycin e- CN-  NADH NAD+ ATP  ADP, Pi Mitochondrial matrix Cyanide (CN-): e- transport inhibitor Oligomycin: ATP synthase inhibitor Dinitrophenol (DNP): Uncoupling agent

14. Cell Cycle + Division Key concepts: G1  S checkpoint via Cyclins, CDKs Tumor suppressors: Rb - phosphorylated by Cyclin D:CDK4 p53 - most common neoplastic mutation 1) Stop cell cycle 2) Promote DNA repair 3) Promote apoptosis

15. Genetics Hardy-Weinberg Equilibrium No mutation No natural selection Random mating No migration For a gene with 2 alleles: (A, a) p + q = 1 p2 + 2pq + q2 = 1 Ex: white irises are a recessive trait determined by a gene with two alleles (A, a). If 16 people out of 100 have white irises, what is the frequency of the carrier state? aa = 16/100 = 0.16 = q2 q = 0.4, p = 0.6 Aa = 2pq = 2 x 0.4 x 0.6 = 0.48

16. Genetics, Pedigree Analysis What type of inheritance? Identify carriers Could this be autosomal dominant? Incomplete Penetrance Key questions: 1) Does the disease skip generations? 2) Does it appear equally in males and females? (transmission patterns)

17. Genetics, Pedigree Analysis • Disease appears in every generation • No male-to-male transmission • 100% male-to-female transmission • For females with disease, 50% of children are affected • X linked dominant

18. 1) Many anti-retroviral drugs combat nascent HIV infection by targeting the critical enzyme necessary for viral replication. Which of the following endogenous enzymes might be expected to experience some degree of cross-reactivity to these therapies? A) DNA Topoisomerase B) RNA Polymerase C) Reverse Transcriptase D) Telomerase

19. 2) Which of the following accurately describes synthesis of the most abundant type of RNA in the cell? A) Synthesized by DNA Pol III in the cytoplasm B) Synthesized by RNA Pol II in the nucleus C) Synthesized by RNA Pol I in the nucleolus D) Synthesized by RNA Pol III in the golgi

20. 3) Which of the following is true regarding the relationship between Prader-Willi and Angelman’s syndrome? A) They are due to a mutation on the X chromosome and represent mosaicism B) They are two types of muscular dystrophy C) They are due to deletion of differentially methylated alleles of the same gene D) They are co-dominant

21. 4) Which of the following effects will be noted when oligomycin is applied to cells undergoing exclusively anaerobic metabolism? A) ATP output will increase B) There will be no change in ATP production C) Electron transport will eventually cease, and a large proton gradient will build up across the inner mitochondrial membrane D) Oxygen consumption will increase

22. 5) Which of the following cellular conditions activate the pyruvate dehydrogenase complex? A) High ATP B) Elevated Acetyl-CoA C) Increased NAD+/NADH ratio D) Decreased Ca2+ E) Reduced Alanine concentration

23. 6) A young infant is found to have prominent epicanthal folds, a simian crease, and a flat facial profile. An abdominal xray demonstrates air on either side of the pyrloric region. This disorder is likely caused by which of the following? A) Maternal infection with a parasite found in cat feces B) An enzyme deficiency C) A trinucleotide repeat expansion D) A meiotic non-disjunction event

24. 7) Hexokinase and glucokinase both catalyze the same reaction (generation of glucose-6-phosphate from glucose). Which of the following best describes the difference between these enzymes? A) Glucokinase has a ubiquitous distribution, whereas hexokinase occurs only in the liver B) Hexokinase has relatively high affinity, whereas glucokinase has low affinity C) Hexokinase is induced by insulin D) Glucokinase has a relatively low capacity, while hexokinase has a high capacity

25. 8) Fermentation occurs under anaerobic conditions to serve which of the following purposes? A) To generate ATP directly in the absence of oxygen B) To generate NADH for use in the electron transport chain C) To regenerate NAD+ so that glycolysis may continue in the absence of oxygen D) To produce Acetyl-CoA for use in the mitochondria

26. 9) A homeless patient appears in the ER visibly intoxicated with alcohol on his breath. He is disoriented as to time and place, on physical exam his ocular cranial nerves do not function normally and he demonstrates an ataxic gate. Which of the following processes is impaired? A) Electron transport chain B) α ketoglutarate decarboxylation C) Lactate formation D) Pyruvate decarboxylation

27. 10) A young child presents with coarse facial features, clouded corneas, and restricted joint movement. Laboratory exams demonstrate a high plasma level of lysosomal enzymes. Which of the following describes the underlying biochemical defect in this disease? A) Impaired glycolysis in skeletal muscle cells B) Defective mannose-6-phosphate addition at the golgi apparatus C) Defective microtubule polymerization in phagocytic cells D) Impaired collagen synthesis in the extracellular compartment

28. 11) A child presents with a history of retardation, aggressive behavior, self-mutilation, and symptomatic gout. Laboratory examination reveals hyperuricemia. What is the correct diagnosis? A) Niemann-Pick disease B) Adenosine deaminase deficiency C) Lesch-Nyhan syndrome D) Osteogenesis imperfecta

29. 12) In Huntington’s disease, the severity of the symptomatology worsens and the age of onset becomes earlier with each successive generation. This is an example of what phenomenon? A) Codominance B) Anticipation C) Pleiotropy D) Loss of heterozygosity

30. 13) A young child presents with a history of progressive neurodegeneration and developmental delay. Upon exam you notice a cherry-red spot on the macula. If this child comes from an Ashkenazi Jewish background, the most likely diagnosis is which of the following: • Pompe’s disease • B) Tay-Sachs disease • C) Fabry’s disease • D) Hurler’s syndrome