Exam 2: Biochemistry

Which two enzymes of the pentose phosphate pathway catalyze production of NADPH?

Passage Leptin signaling is vital for maintaining adequate body mass and preventing insulin resistance.  When energy stores are abundant, adipocytes produce leptin to stimulate fatty acid oxidation and glucose utilization.  One result of leptin signaling in skeletal muscle cells is the phosphorylation and activation of adenosine monophosphate-activated protein kinase (AMPK).  AMPK facilitates the breakdown of triglycerides by decreasing the activity of acetyl-coenzyme A carboxylase (ACC); this leads to downregulation of fatty acid synthesis and upregulation of β-oxidation.Fatty acid oxidation has been studied by analyzing the effect of leptin on activation of AMPK and ACC in soleus muscles.  To identify leptin's mode of action, injections were administered to transgenic mice via the intravenous (1 mg/kg i.v.) and intrahypothalamic (1 ng i.h.p [faster drug delivery]) routes.  Phentolamine (PH), an α-adrenergic receptor ligand that binds to the same receptor as leptin, was administered concurrently in some of the mice.  Signaling to ACC from the citric acid cycle was also examined through the addition of citrate. Figure 1  AMPK activity measured in soleus muscle injected with leptin and PH via i.v. and i.h.p.Table 1  ACC Activity in Soleus Muscle Exposed to Leptin Delivered via i.v. and i.h.p Injections in vivo Adapted from Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature. 2002;415(6869):339-43. -In skeletal muscle cells, leptin resistance would lead to decreased concentrations of:

Medium-chain acyl-CoA dehydrogenase (MCAD) is an enzyme involved in β-oxidation, and is encoded by the autosomal ACADM gene.  MCAD deficiency is caused by a mutation in ACADM, and results in impaired ability to digest medium-chain fatty acids for energy, and in their subsequent accumulation.  Which of the following interventions would best relieve the negative effects of MCAD deficiency?

If a stretch of double-stranded DNA has a total of 560 nucleotides and 159 adenine nucleotides, what is the nucleotide composition of this DNA molecule?  It has:

Passage Glycolysis and gluconeogenesis are tightly regulated, opposing metabolic pathways that help control blood glucose levels.  Glycolysis converts glucose to two pyruvate molecules, whereas gluconeogenesis consumes 6 ATP equivalents to convert two pyruvate molecules back to glucose.  When glycolysis is upregulated, gluconeogenesis is downregulated, and vice versa.As shown in Figure 1, glycolysis and gluconeogenesis in the liver are largely regulated by the allosteric action of the small molecule fructose-2,6-bisphosphate (F2,6BP) on the enzymes phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F1,6BPase).  PFK-1 is a kinase that uses ATP to phosphorylate fructose-6-phosphate (F6P) in an irreversible step of glycolysis, forming fructose-1,6-bisphosphate (F1,6BP) and ADP.  During gluconeogenesis, F1,6BPase removes the phosphate group by hydrolysis. Figure 1  Activities of (A) PFK-1 and (B) F1,6BPase in the presence (solid lines) and absence (dashed lines) of F2,6BPA bifunctional enzyme that contains a phosphofructokinase-2 (PFK-2) domain and a fructose-2,6-bisphosphatase (F2,6BPase) domain controls F2,6BP levels in the liver.  The PFK-2 domain converts F6P to F2,6BP, and the F2,6BPase domain converts F2,6BP back to F6P.  When blood glucose levels are low, the enzyme becomes phosphorylated.  This phosphorylation event simultaneously activates the F2,6BPase domain and inactivates the PFK-2 domain.  Under high blood glucose conditions, the enzyme becomes dephosphorylated, activating the PFK-2 domain and inactivating the F2,6BPase domain. -The information in the passage and the data in Figure 1 support the conclusion that when glycolysis is active, the bifunctional PFK-2/F2,6BPase enzyme is:

The electron transport chain results in the generation of:

Passage The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride ion channel involved in the production of mucus, sweat, and digestive fluids.  CFTR is composed of five domains (Figure 1):  Two transmembrane domains (TMD1 and TMD2), two nucleotide binding domains (NBD1 and NBD2), and one regulatory domain (R domain). Figure 1  Schematic diagram of CFTR domainsThe NBD domains regulate CFTR activity by binding ATP, which causes a conformational change in the TMD domains.  This change allows chloride ions to leave cells by passively crossing the membrane down their concentration gradient.  The R domain regulates activity further through dynamic phosphorylation at several positions.  The movement of chloride ions facilitates the movement of water out of the cell by osmotic pressure and helps keep secretions thin.Cystic fibrosis (CF) arises from recessively inherited mutations in the CFTR gene.  The most common mutation is a three-base-pair deletion that removes a phenylalanine residue at position 508 in NBD1 (ΔF508).  This mutation causes the protein to fold incorrectly, making it more susceptible to degradation by proteases.  The resulting decrease in CFTR abundance inhibits chloride ion transport, leading to thickening of normally thin secretions and increased risk of life-threatening pulmonary infections. -According to the information in the passage, which statement best describes the role of ATP in CFTR activity?  ATP:

A protein consisting of domains A, B, and C binds a ligand.  Which experimental result would provide the best evidence that domain A is involved in binding and domains B and C are not?

Passage Many cephalopods can change color to blend in with their surroundings by altering the conformations of particular proteins within their skin cells.  The reflectin proteins play a major role in this camouflage mechanism.  Reflectins are a group of intrinsically disordered proteins that do not adopt clearly defined secondary or tertiary structures.  Under certain conditions reflectins aggregate, allowing cells to reflect various wavelengths of light.Skin cells from the squid Doryteuthis opalescens were cultured and three reflectins-DoRA, DoRB, and DoRC-were purified and analyzed to determine amino acid composition.  They were found to contain an abundance of arginine and serine but relatively few cysteine, leucine, isoleucine, valine, phenylalanine, or tryptophan residues.  Figure 1 shows the relative hydrophobicity of each reflectin variant as a function of amino acid position. Figure 1  Hydrophobicity plots of three reflectins isolated from D. opalescens.  (Note: Negative numbers indicate hydrophilic regions.)In D. opalescens skin cells (pH ≈ 7.8), reflectins aggregate in response to acetylcholine (ACh), which causes a signal cascade that changes the phosphorylation state of the proteins.  Cells were incubated with or without ACh, and DoRA and DoRB were purified and assessed for phosphorylation by mass spectrometry.  DoRA became phosphorylated in the presence of ACh whereas DoRB became dephosphorylated.  After examining the isoelectric points of unmodified DoRA and DoRB, as shown in Figure 2, scientists proposed that reflectins aggregate upon charge neutralization. Figure 2  Isoelectric focusing of DoRA and DoRB Adapted from Demartini DG, Izumi M, Weaver AT, Pandolfi E, Morse DE.  Structures, Organization, and Function of Reflectin Proteins in Dynamically Tunable Reflective Cells.  J Biol Chem. 2015;290(24):15238-49. -In the absence of ACh, which of the following bond types best characterizes the structure of reflectins?

Passage The HIV-derived transactivator of transcription (Tat) is a short peptide with the sequence RKKRRQRRRPPQ.  When paired with liposomes (ie, synthetic vesicles) composed of cationic lipids, Tat can transport large molecules across cell membranes and has been studied as a vector for gene therapy.  However, Tat delivers cargo to cells with relatively low efficiency.  Researchers hypothesized that a Tat homodimer could yield stronger DNA binding than the monomer, which may improve the efficiency of DNA delivery.To study this hypothesis, Tat and two variant peptides (Variants 1 and 2) were produced by solid-phase peptide synthesis, in which the growing peptide chain is immobilized on polystyrene beads.  Unlike biological systems, solid-phase synthesis assembles peptides from the C-terminus to the N-terminus, as shown in Figure 1. Figure 1  Simplified schematic of one round of solid-phase peptide synthesis.  The protecting group is removed during deprotection.  The process is repeated until the peptide is complete.Variants 1 and 2 contained a C-terminal and an N-terminal cysteine residue, respectively.  Each variant was allowed to dimerize by disulfide bond formation.  Peptide-DNA binding interactions were then studied by mixing varying amounts of each peptide with plasmid DNA encoding luciferase protein (pLuc).  The resulting peptide-DNA complexes were run on agarose gels, with results displayed in Figure 2. Figure 2  Agarose gel results for pLuc plasmid exposed to varying amounts of each peptideEqual concentrations of each type of peptide-DNA complex were then incubated with cationic liposomes and exposed to cultured cells for 24 hours.  The cells were then assessed for luciferase activity.  Greater luciferase activity indicated more efficient delivery of the pLuc plasmid.  The results are shown in Figure 3. Figure 3  Plasmid delivery efficiency of three peptides, as measured by induced luciferase activity Adapted from Lee S-J, Yoon S-H, Doh K-O. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond. J Microbiol Biotechnol. 2011;21(8):802-807. -The researchers observed that the last three amino acids added to the Tat monomer during solid-phase peptide synthesis required more time for linkage than the first several amino acids added.  Which of the following best explains this observation?

Passage Glycolysis and gluconeogenesis are tightly regulated, opposing metabolic pathways that help control blood glucose levels.  Glycolysis converts glucose to two pyruvate molecules, whereas gluconeogenesis consumes 6 ATP equivalents to convert two pyruvate molecules back to glucose.  When glycolysis is upregulated, gluconeogenesis is downregulated, and vice versa.As shown in Figure 1, glycolysis and gluconeogenesis in the liver are largely regulated by the allosteric action of the small molecule fructose-2,6-bisphosphate (F2,6BP) on the enzymes phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F1,6BPase).  PFK-1 is a kinase that uses ATP to phosphorylate fructose-6-phosphate (F6P) in an irreversible step of glycolysis, forming fructose-1,6-bisphosphate (F1,6BP) and ADP.  During gluconeogenesis, F1,6BPase removes the phosphate group by hydrolysis. Figure 1  Activities of (A) PFK-1 and (B) F1,6BPase in the presence (solid lines) and absence (dashed lines) of F2,6BPA bifunctional enzyme that contains a phosphofructokinase-2 (PFK-2) domain and a fructose-2,6-bisphosphatase (F2,6BPase) domain controls F2,6BP levels in the liver.  The PFK-2 domain converts F6P to F2,6BP, and the F2,6BPase domain converts F2,6BP back to F6P.  When blood glucose levels are low, the enzyme becomes phosphorylated.  This phosphorylation event simultaneously activates the F2,6BPase domain and inactivates the PFK-2 domain.  Under high blood glucose conditions, the enzyme becomes dephosphorylated, activating the PFK-2 domain and inactivating the F2,6BPase domain. -What effect does glucagon most likely have on F2,6BP synthesis in the liver?  Glucagon:

Which statement accurately describes a substrate in the presence of an enzyme operating at V_max?  Only when the enzyme operates at V_max is the substrate:

Passage Spinocerebellar ataxia 3 (SCA3) is a neurodegenerative disease that manifests as a progressive decrease in coordination of limbs and frequently results in impaired speech and eye coordination.  It is associated with extension of the polyglutamine (polyQ) region of the ataxin-3 protein.  The polyQ region is encoded by a series of consecutive CAG codons known as poly-CAG repeats.  These regions vary in length from one person to another, ranging from as few as 10 to as many as 80 repeats.  SCA3 has been reported in patients with polyQ regions exceeding ~55 repeats, and increased polyQ length correlates with earlier onset of the disease and greater clinical severity.Circular dichroism (CD) is a method of assessing protein secondary structure.  It measures a molecule's ability to absorb left- and right-handed circularly polarized light of varying wavelengths.  The absorption difference is known as "ellipticity," denoted by Δε.  Alpha-helices have a maximum Δε at 190 nm, and beta-sheets have a maximum Δε at 200 nm.CD results show that as the length of the ataxin-3 polyQ region increases, alpha-helical nature is lost and the protein aggregates, ultimately precipitating out of solution and depositing in brain and other tissues.  Furthermore, as proteins aggregate, they form beta-sheets.  These beta-sheets are in the parallel orientation, and each sheet can interact with sheets in other proteins to form long, insoluble structures known as amyloid fibers (Figure 1). Figure 1  Depiction of the ataxin-3 polyQ region shifting from alpha-helical (cylinders) to beta-sheet (arrows) structure and aggregatingGlutamine can be encoded by both CAG and CAA codons.  Researchers investigated survival rates in fruit flies expressing ataxin-3 with polyQ regions of equal length encoded by either CAG-CAG or CAA-CAG repeats (Table 1).  The protein products had identical amino acid sequences.Table 1  Fruit Fly Survival Rates Relative to Ataxin-3 Expression Adapted from Bevivino AE, Loll PJ. An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel beta -fibrils. Proc Natl Acad Sci USA. 2001;98(21):11955-60. -Which statements regarding ataxin-3 parallel beta-sheets are true?Short sequences called beta-turns link one strand to the next.The N-termini of each strand are aligned.The adjacent backbones hydrogen bond with each other.

Passage Leptin signaling is vital for maintaining adequate body mass and preventing insulin resistance.  When energy stores are abundant, adipocytes produce leptin to stimulate fatty acid oxidation and glucose utilization.  One result of leptin signaling in skeletal muscle cells is the phosphorylation and activation of adenosine monophosphate-activated protein kinase (AMPK).  AMPK facilitates the breakdown of triglycerides by decreasing the activity of acetyl-coenzyme A carboxylase (ACC); this leads to downregulation of fatty acid synthesis and upregulation of β-oxidation.Fatty acid oxidation has been studied by analyzing the effect of leptin on activation of AMPK and ACC in soleus muscles.  To identify leptin's mode of action, injections were administered to transgenic mice via the intravenous (1 mg/kg i.v.) and intrahypothalamic (1 ng i.h.p [faster drug delivery]) routes.  Phentolamine (PH), an α-adrenergic receptor ligand that binds to the same receptor as leptin, was administered concurrently in some of the mice.  Signaling to ACC from the citric acid cycle was also examined through the addition of citrate. Figure 1  AMPK activity measured in soleus muscle injected with leptin and PH via i.v. and i.h.p.Table 1  ACC Activity in Soleus Muscle Exposed to Leptin Delivered via i.v. and i.h.p Injections in vivo Adapted from Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature. 2002;415(6869):339-43. -Fatty acids must be activated prior to degradation by a ligase enzyme.  Which of the following reactions is catalyzed by a ligase?

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation.  The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization.  Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs.  These hydroxylated residues serve as markers for the ubiquitin-proteasome system.  Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs.  Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1  Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FH⁺) and fumarate-deficient (FH⁻) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2  HIF-1a in FH⁺ and FH⁻ cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -Why did researchers test for the α-tubulin protein along with HIF-1α?

Passage Antimicrobial peptides are an important component of innate immunity.  Since their discovery in 1939, over 5,000 peptides with antimicrobial properties have been isolated from organisms in every domain of life.  They have demonstrated activity against enveloped viruses, bacteria, fungi, and even tumor cells and have recently gained interest for their potential in fighting antibiotic-resistant bacteria.  Most known antimicrobial peptides are positively charged, but some with negative charges have also been discovered.Frogs are known to secrete a variety of antimicrobial peptides as a major source of immunity.  Secretions from a new species of frog were collected and the peptides were separated by cation exchange chromatography.  Four of the isolated peptides demonstrated strong antibacterial activity and were characterized by isoelectric focusing and sequencing: Figure 1  Results of isoelectric focusing of four antimicrobial peptidesTable 1  Amino Acid Compositions of Four Antimicrobial Peptides Adapted from Wang Z, Wang G. APD: the Antimicrobial Peptide Database. Nucleic Acids Res. 2004;32(Database issue):D590-2. -One of the challenges of antimicrobial peptides is their short physiological half-life.  To solve this problem, some researchers have begun investigating synthetic antimicrobial peptides composed of D-amino acids.  Why might these peptides have an increased half-life in the body relative to natural antimicrobial peptides?

Which of the following correctly describes the stereospecific formation of a citric acid cycle intermediate?

Passage Spinocerebellar ataxia 3 (SCA3) is a neurodegenerative disease that manifests as a progressive decrease in coordination of limbs and frequently results in impaired speech and eye coordination.  It is associated with extension of the polyglutamine (polyQ) region of the ataxin-3 protein.  The polyQ region is encoded by a series of consecutive CAG codons known as poly-CAG repeats.  These regions vary in length from one person to another, ranging from as few as 10 to as many as 80 repeats.  SCA3 has been reported in patients with polyQ regions exceeding ~55 repeats, and increased polyQ length correlates with earlier onset of the disease and greater clinical severity.Circular dichroism (CD) is a method of assessing protein secondary structure.  It measures a molecule's ability to absorb left- and right-handed circularly polarized light of varying wavelengths.  The absorption difference is known as "ellipticity," denoted by Δε.  Alpha-helices have a maximum Δε at 190 nm, and beta-sheets have a maximum Δε at 200 nm.CD results show that as the length of the ataxin-3 polyQ region increases, alpha-helical nature is lost and the protein aggregates, ultimately precipitating out of solution and depositing in brain and other tissues.  Furthermore, as proteins aggregate, they form beta-sheets.  These beta-sheets are in the parallel orientation, and each sheet can interact with sheets in other proteins to form long, insoluble structures known as amyloid fibers (Figure 1). Figure 1  Depiction of the ataxin-3 polyQ region shifting from alpha-helical (cylinders) to beta-sheet (arrows) structure and aggregatingGlutamine can be encoded by both CAG and CAA codons.  Researchers investigated survival rates in fruit flies expressing ataxin-3 with polyQ regions of equal length encoded by either CAG-CAG or CAA-CAG repeats (Table 1).  The protein products had identical amino acid sequences.Table 1  Fruit Fly Survival Rates Relative to Ataxin-3 Expression Adapted from Bevivino AE, Loll PJ. An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel beta -fibrils. Proc Natl Acad Sci USA. 2001;98(21):11955-60. -Which of the following could increase the stability of the beta-conformation of long polyglutamine regions?

Consider the following reaction: Which parameter would NOT be affected by the addition of phosphoglycerate mutase?

Passage The liver plays a central role in maintaining blood glucose homoeostasis.  It contains the highest concentration of glycogen of any organ in the body and is one of few organs that can regenerate glucose from metabolic byproducts.  The liver can replenish glucose in muscle and brain tissue during times of fasting, and it can help prevent lactate accumulation during intense physical activity.Von Gierke disease is a rare autosomal recessive disorder that arises from inactivating mutations in the liver enzyme glucose 6-phosphatase (G6Pase).  It affects roughly 1 in 100,000 individuals, and the resulting loss of both hormonal sensitivity and enzymatic activity leads to hypoglycemia (low blood sugar).  Von Gierke can also cause an increase in blood acidity concurrent with lactate build-up.  Patients typically become symptomatic shortly after birth with convulsions, hyperventilation, and tremors.  Left untreated, patients may develop gout, osteoporosis, and life-threatening complications such as kidney failure and liver tumors.In an effort to examine the biochemical consequences of this disorder, scientists generated a genetically engineered mouse model with liver G6Pase expression under the control of the Tet-Off system at both alleles.  Under this system, addition of the small molecule tetracycline in the water source of the mouse rapidly shuts off G6Pase gene expression and therefore can recapitulate von Gierke disease.  Blood glucose and pH levels were measured daily in two groups of genetically engineered mice over 20 days: group A mice received no tetracycline and group B mice were given tetracycline starting at day 10.  All mice were fed identical low-sugar diets throughout the experiment. Adapted from Froissart R, Piraud M, Boudjemline AM, et al. Glucose-6-phosphatase deficiency. Orphanet J Rare Dis. 2011;6:27. -Which of the following graphs best reflects the expected average blood glucose and pH levels over time for group B mice?