Exam 2: Biochemistry

Passage Diabetic nephropathy (kidney disease) is characterized in part by a significant decrease in the glomerular filtration rate.  Podocytes are differentiated cells that surround the glomerular capillaries and counteract the pressure of blood filtration.  Hyperglycemia (elevated blood glucose) and capillary hypertension (high blood pressure), both of which are common in diabetic patients, can subject podocytes to mechanical stress.  This mechanical stress has been shown to regulate glucose uptake by podocytes, possibly by altering the production of glucose transport (GLUT) proteins.Researchers investigating GLUT2 and GLUT4 expression in rat podocytes measured the impact of mechanical stress on glucose uptake.  Cells were grown for 4 hours on membranes exposed to atmospheric (atm) or high air pressure (HP) to mimic mechanical stress.  Antibodies specific to GLUT2 and GLUT4 were used to visualize the expression of each under normal glucose (NG) and hyperglycemic (HG) conditions, and to assess the effect of mechanical stress on expression.  The uptake of a radiolabeled glucose analogue, [³H]-2-deoxy-D-glucose (3H-2DG), was then determined.  The quantity of 3H-2DG transported by each GLUT protein was directly proportional to the cell surface expression. Figure 1  Cell surface expression of GLUT transporters in response to mechanical stress and varying glucose concentration.In vivo studies have shown that transport via GLUT2 is noninducible and depends only on extracellular glucose concentrations.  However, GLUT4-mediated uptake is induced by insulin binding and occurs under high glucose conditions when insulin is released.  The standard K_m values for GLUT2 and GLUT4 are 19.4 and 4.3 mM, respectively, at normal blood glucose levels after a meal (~7.8 mM).  The data shown in Figure 2 represent the combined kinetic properties of both transporters. Figure 2  Plot of glucose uptake in podocytes with and without mechanical stress. Adapted from Lewko, B., Bryl, E., Witkowski, J. M., Latawiec, E., Angielski, S., & Stepinski, J. (2005). Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes. Nephrology Dialysis Transplantation, 20(2), 306-311. -Researchers inverted the data shown in Figure 2 and observed that the values fit a hyperbolic curve.  What conclusion is best supported by their observation?

Passage Myalgic encephalopathy (ME), or chronic fatigue syndrome (CFS), is a physiological disease that affects 0.15% of the human population.  Compared with healthy individuals, persons with ME experience reduced phospholipid levels, muscular degeneration, urinary urgency, and other symptoms that contribute to a lower quality of life.  Studies in resting subjects have yielded inconsistent data and failed to identify a unique metabolic defect in patients with ME.Analyzing the blood plasma of male and female patients with ME, physicians have observed that male patients display elevated levels of 3-methylhistidine, a marker of protein catabolism.  Researchers expanded this finding by measuring the serum concentration of amino acids in blood samples obtained from nonfasting ME patients and healthy controls.  Amino acids were classified into separate categories according to their metabolic intermediates.Table 1  Classification of Amino Acids in Blood Plasma Figure 1  Serum concentrations of amino acids in patients with ME and healthy controls (HC)Next, researchers hypothesized that the inhibition of pyruvate dehydrogenase (PDH) and impairment of fatty acid oxidation were responsible for the fluctuations in amino acid levels.  PDH function and lipid metabolism were studied in peripheral blood mononuclear cells (PBMCs) by measuring the gene expression of proteins within regulatory pathways.  Researchers monitored the mRNA levels of the inhibitor pyruvate dehydrogenase kinase 1 (PDK1) and of sirtuin 4 (SIRT4), which removes lipoic acid groups from the PDH complex.  They also examined lipid metabolism by measuring the mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), a transcription factor that upregulates the uptake of fatty acids.  The mRNA level of each protein in PBMCs of patients with ME was evaluated against healthy controls, as shown in Figure 2. Figure 2  Gene expression of PDK1, SIRT4, and PPARα in PBMCs Adapted from Fluge Ø, Mella O, Bruland O, et al.. JCI Insight. 2016;1(21):e89376. -Assuming that 3-methylhistidine is the only indicator of protein catabolism, which observation would best support the difference in the metabolism of amino acids in male and female patients with ME?

Passage Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis.  This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin.  Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1). Figure 1  Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins.  Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1.  The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1  Composition of cis-Complex Zero Ionic Layer An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF), which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake.  Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin.  After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting.  Results are shown in Figure 2. Figure 2  α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5):2213-7. -Wild-type syntaxin (Synt-WT) and mutant syntaxins containing a deletion of the N-terminal sequence (SyntΔA) or C-terminal sequence (SyntΔB) were separated and incubated with α-SNAP.  After washing away unbound molecules, which of the following is the most likely result of protein immunoblotting for α-SNAP?

Passage Whole grains have been shown to reduce the risk of cardiovascular disorders.  A team of scientists discovered that the compound 5-aminovaleric acid betaine (5-AVAB) was present at high levels in cardiomyocytes of mice given a diet rich in whole grains and decided to investigate its role in metabolism.  Preliminary evidence indicated that 5-AVAB did not alter the citric acid cycle in these cardiomyocytes, but that it did alter fatty acid metabolism.The plasma membrane transport protein organic cation/carnitine transporter 2 (OCTN2) transports extracellular L-carnitine into the cell against its concentration gradient using the energy provided by the simultaneous transport of Na⁺ into the cell.  Cellular L-carnitine is required for the transport of fatty acids from the cytosol into the mitochondria.  Based on the structures of 5-AVAB and L-carnitine (Figure 1), scientists hypothesized that 5-AVAB may interfere with OCTN2-mediated transport of L-carnitine. Figure 1  Structures of L-carnitine and 5-AVABTo test their hypothesis, scientists incubated mouse cardiomyocytes with various concentrations of 5-AVAB for 24 hours.  Cells were then provided with the fatty acid palmitate for energy and were deprived of all other energy sources.  The cardiomyocytes were also given 50 μM L-carnitine (endogenous extracellular concentration) or 2 mM L-carnitine, and the rate of oxygen consumption was monitored (Figure 2). Figure 2  Oxygen consumption rate of cardiomyocytes at various concentrations of 5-AVAB and L-carnitineIn a follow-up experiment, the scientists used siRNA to silence expression of OCTN2 and found that intracellular L-carnitine levels were similarly depleted in siRNA-treated cells as in cells treated with 100 μM 5-AVAB. Adapted from Kärkkäinen O, Tuomainen T, Koistinen V, et al. Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes. Sci Rep. 2018;8(1):13036. -Under which of the conditions tested in Figure 2 would cardiomyocytes have the highest ADP/ATP ratio?

Passage Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis.  This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin.  Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1). Figure 1  Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins.  Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1.  The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1  Composition of cis-Complex Zero Ionic Layer An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF), which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake.  Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin.  After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting.  Results are shown in Figure 2. Figure 2  α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5):2213-7. -Which of the following amino acid substitutions is most likely to interrupt the interactions of the zero ionic layer?

An enzyme kinetics assay reveals that V_max = 100 nmol/s and K_m = 10 μM.  For this assay, what substrate concentration is required for a reaction rate V₀ of 75 nmol/s?

Passage DNA polymerization is one of the most conserved mechanisms of genome replication.  Synthesis of a complete DNA strand requires a template, primers, a polymerase enzyme, and sufficient deoxyribonucleotide triphosphates (dNTPs).  The DNA polymerase enzyme binds consecutive base pairs on the template strand and extends the double helix by adding dNTPs to the primer.  The amino acid residues in the active site of DNA polymerase form hydrogen bonds with Watson-Crick donors and acceptors on incoming DNA nucleotides to facilitate base pairing.The formation of the DNA double helix creates opposing changes in entropy and enthalpy.  Favorable bonding interactions via hydrogen bonds during Watson-Crick base pairing results in negative enthalpy, and restricted rotation and flexibility of the DNA backbone generates negative entropy.  Scientists hypothesize that hydrogen bonding between bases not only stabilizes the double helix but is also crucial for selective and efficient replication.Analogs that are similar in size and shape to naturally occurring bases can be used to determine the influence of hydrogen bonding on base pair selectivity.  To mimic the structure of deoxythymidine triphosphate (dTTP), researchers synthesized dNTP derivatives of difluorotoluene (dFTP), a nonpolar analog that lacks Watson-Crick hydrogen bonding.  Klenow fragment (KF) polymerase, which has 3′-5′ but not 5′-3′ exonuclease activity, was incubated with a mixture of DNA template, primers, and dNTPs, including dFTP derivatives.  The efficiency of dFTP and natural dTTP nucleotide incorporation into a growing primer strand by KF is shown in Figure 1. Figure 1  Template-specific selection of dFTP and dTTP by the KF enzyme Adapted from Moran S, Ren RX, Kool ET. A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity. Proc Natl Acad Sci USA. 1997;94(20):10506-11. -The experimental results described in the passage are inconsistent with which hypothesis?

Passage Physical activity produces an increased energy requirement in the muscles as well as in the brain.  During periods of intense exercise, glycogenolysis is activated in muscles to provide them with the energy needed for contractions, and research indicates that astrocytes also consume glycogen at increased levels during exercise.  It is hypothesized that during periods of physical exertion, astrocytes convert glycogen to lactate, which is then used as a fuel source in neurons.Monocarboxylate transporters (MCTs) are responsible for the movement of lactate across membranes, and MCT2 is expressed at increased levels in neuronal cells during exercise.  Scientists proposed that lactate is secreted from astrocytes and taken up by neurons through MCT2.  Lactate is then converted to pyruvate and enters the mitochondria, where it is further metabolized.  To test their predictions, scientists examined the effect of exercise on glycogen metabolism in rodents.Experiment 1Rats were either allowed to rest or exercised to exhaustion.  The levels of several metabolites were then quantified in both the plantaris muscle and the hippocampus. Figure 1  Levels of several metabolites in plantaris muscle (A) and hippocampus (B) from rats at rest or after exercising to exhaustionExperiment 2Rats were injected with 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) or saline, and glycogen and lactate levels were measured in the hippocampus of rats at rest or after exercise to exhaustion. Figure 2  Levels of glycogen (A) and lactate (B) in rats injected with DAB or saline, after rest or upon exhaustion Adapted from Matsui T, Omuro H, Liu Y-F, et al. Astrocytic glycogen-derived lactate fuels the brain during exhaustive exercise to maintain endurance capacity. Proceedings of the National Academy of Sciences. 2017;114(24):6358-6363. -DAB most likely inhibits which reaction?

Passage Myalgic encephalopathy (ME), or chronic fatigue syndrome (CFS), is a physiological disease that affects 0.15% of the human population.  Compared with healthy individuals, persons with ME experience reduced phospholipid levels, muscular degeneration, urinary urgency, and other symptoms that contribute to a lower quality of life.  Studies in resting subjects have yielded inconsistent data and failed to identify a unique metabolic defect in patients with ME.Analyzing the blood plasma of male and female patients with ME, physicians have observed that male patients display elevated levels of 3-methylhistidine, a marker of protein catabolism.  Researchers expanded this finding by measuring the serum concentration of amino acids in blood samples obtained from nonfasting ME patients and healthy controls.  Amino acids were classified into separate categories according to their metabolic intermediates.Table 1  Classification of Amino Acids in Blood Plasma Figure 1  Serum concentrations of amino acids in patients with ME and healthy controls (HC)Next, researchers hypothesized that the inhibition of pyruvate dehydrogenase (PDH) and impairment of fatty acid oxidation were responsible for the fluctuations in amino acid levels.  PDH function and lipid metabolism were studied in peripheral blood mononuclear cells (PBMCs) by measuring the gene expression of proteins within regulatory pathways.  Researchers monitored the mRNA levels of the inhibitor pyruvate dehydrogenase kinase 1 (PDK1) and of sirtuin 4 (SIRT4), which removes lipoic acid groups from the PDH complex.  They also examined lipid metabolism by measuring the mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), a transcription factor that upregulates the uptake of fatty acids.  The mRNA level of each protein in PBMCs of patients with ME was evaluated against healthy controls, as shown in Figure 2. Figure 2  Gene expression of PDK1, SIRT4, and PPARα in PBMCs Adapted from Fluge Ø, Mella O, Bruland O, et al.. JCI Insight. 2016;1(21):e89376. -Extended periods of physical exertion in patients with ME result in the conversion of glycine and cysteine to which of the following compounds?

An enzyme generates a Lineweaver-Burk plot with a slope of m.  If the enzyme's Michaelis constant K_m is known, which expression yields the maximum velocity V_max of the enzyme-catalyzed reaction?

Passage The recent advent of multidrug-resistant Mycobacterium tuberculosis (Mtb) has led to the search for novel agents to combat tuberculosis.  Mtb secretes over 70 serine proteases that may be used as viable drug targets.  Hydrolase important for pathogenesis 1 (Hip1) is a serine protease found in Mtb with proteolytic activity that is required for dampening the host inflammatory response.  Given the role of Hip1 in Mtb virulence, analyzing Hip1 substrate specificity is a critical step in designing selective inhibitors for effective tuberculosis treatment.Fluorogenic substrates are nonfluorescent compounds that release a fluorescent reporter group when acted upon by an enzyme.  Such substrates are useful biomolecular imaging tools that allow monitoring of enzymatic activity in vitro and in vivo.  A large library of peptides was screened against Hip1, and enzymatic cleavage was analyzed using mass spectrometry to facilitate the design of fluorogenic substrates (Figure 1). Figure 1  Schematic design of Hip1 fluorogenic substrates.  P1, P2, P3, and P4 in Group 1 represent the positions of variable amino acids in the peptide.  Group 2 depicts the fluorescent compound released on Hip1 cleavage.Researchers reported Michaelis-Menten kinetic parameters for substrates from two peptide families: the WKLL substrates and the CSL substrates.Table 1  Kinetic Parameters of Fluorogenic Substrates for Mtb Hip1 Adapted from Lentz CS, Ordonez AA, Kasperkiewicz P, et al. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect Dis. 2016;2(11):807-815. -In further experiments researchers found that bacteria lacking Hip1 (knockout bacteria) were able to cleave WKLL-ACC but not the CSL substrates.  How might these results be explained?

Fatty acid oxidation and synthesis occur in which subcellular compartments, respectively?

Under physiological conditions, peptide bond formation and degradation both require enzymes, but only formation requires coupling to GTP hydrolysis.  Based on this information, peptide bonds under physiological conditions are:

Passage Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis.  This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin.  Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1). Figure 1  Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins.  Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1.  The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1  Composition of cis-Complex Zero Ionic Layer An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF), which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake.  Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin.  After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting.  Results are shown in Figure 2. Figure 2  α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5):2213-7. -Which amino acid is LEAST likely to be found in normally functioning binding domains of t-SNARE and v-SNARE proteins?

Passage NDPK is an enzyme in budding yeast that uses ATP to phosphorylate nucleotide diphosphates (NDPs and dNDPs), yielding the triphosphate form of each (NTPs and dNTPs).  dNTPs are necessary for DNA synthesis, yet yeast cells that lack NDPK grow and divide at the same rate as wild-type yeast.  Therefore, triphosphate generation is most likely carried out by one or more other enzymes.The enzyme TMPK converts deoxythymidine monophosphate (dTMP) to the diphosphate form (dTDP) by transferring the γ-phosphate of ATP to dTMP.  Yeast cells that lack TMPK activity cannot synthesize DNA and become arrested during replication.  It was proposed that TMPK may partially compensate for NDPK loss by phosphorylating dTDP as well as dTMP.To better understand the role of TMPK in nucleotide phosphorylation, wild-type TMPK and the inactive E75K variant were purified by affinity chromatography, and the following experiments were performed.Experiment 1After purification, a sample containing 3 μg of protein was added to a reaction mixture containing radiolabeled nucleotide monophosphates (NMPs or dNMPs) and unlabeled ATP.  The reaction proceeded for 30 minutes, and products were separated by thin-layer chromatography (TLC) on a PEI-cellulose plate.  PEI-cellulose separates nucleotides by charge (Figure 1), and migration distance decreases with increasing negative charge.  Spots were visualized by autoradiography, and it was determined that 0.30 nmol of dTMP and 0.15 nmol of uridine monophosphate (UMP) became phosphorylated, respectively. Figure 1  TLC analysis of nucleotide monophosphates after exposure to TMPK and ATP.  (Note: Different nucleotides with the same phosphorylation level migrate different distances.)Experiment 2The experiment was repeated using radiolabeled dTDP as the starting material.  The reaction proceeded in buffer only, in the presence of E75K TMPK, or in the presence of wild-type TMPK.  Products were separated by TLC and visualized (Figure 2). Figure 2  TLC analysis of dTDP phosphorylation with buffer only (Lane 1), E75K TMPK (Lane 2), or wild-type TMPK (Lane 3) Adapted from Chien CY, Chen BR, Chou CK, Sclafani RA, Su JY. The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett. 2009;583(13):2281-6. -Which statement about TMPK specificity is supported by Figure 1?

Passage Aldehyde dehydrogenase 2 (ALDH2) is essential for the oxidation of toxic aldehydes, and uses NAD⁺ as the oxidizing agent.  Mitochondrial ALDH2 exists as a 220-kD homotetrameric protein containing a catalytic, nucleotide-binding, and coenzyme-binding domain.  The most prevalent ALDH2 mutation (ALDH2*2) involves an E to K (E478K) substitution in the catalytic domain that leads to decreased enzymatic activity.Scientists hypothesize that Asian populations have higher rates of ALDH2*2 incidence.  The properties of ALDH2 variants in Caucasian and Japanese populations were analyzed in the following experiments.Experiment 1Mitochondrial extracts from both populations were combined in a single mixture and separated by native gel electrophoresis.  Proteins 210-230 kD in size were removed from the agarose gel and further isolated using affinity columns with antibodies designed to separate wild-type ALDH2 from ALDH2*2 by binding the catalytic domain of the wild-type but not the mutant protein.  Concentrated eluates E1 and E2 were produced by washing the affinity columns with buffers that remove unbound proteins and dislodge bound proteins from antibodies, respectively (Figure 1). Figure 1  Purification of ALDH2 proteinsALDH2 activity in each eluate was quantified by adding acetaldehyde and NAD⁺, and then monitoring the rate of NADH production.  The results are shown in Figure 2. Figure 2  Enzymatic activity of E1 and E2 eluatesExperiment 2Researchers predict that mutations at conserved amino acid sites containing S or T may prevent ALDH2 from being phosphorylated by protein kinase C epsilon (εPKC).  The activity of wild-type ALDH2 (WT) and ALDH2 mutants with substitutions at positions T185 and S279 in the presence or absence of εPKC are summarized in Table 1.Table 1  Effect of εPKC on ALDH2 Variants Adapted from Chen CH, Ferreira JC, Gross ER, Mochly-rosen D. Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev. 2014;94(1):1-34. -Based on Table 1, which pair of amino acid substitutions in ALDH2 would most likely result in the highest NADH production in the absence of εPKC?

Passage The innate immune system relies heavily on the phagocytic action of neutrophils, mobile white blood cells that engulf and degrade bacteria and viruses as the first line of host defense against invading pathogens.  One mechanism for eliminating viruses engulfed in the phagosome involves the degradation of viral proteins by neutrophil serine proteases (NSPs).NSPs, which form part of the chymotrypsin family of serine proteases, cleave viral proteins using a catalytic triad relay system.  Originally discovered in granules released by neutrophils, NSP4 is a novel monomeric serine protease that has been studied to elucidate the catalytic mechanism of NSPs.  Prior experimental data suggest that NSP4 alters its tertiary structure upon substrate binding and cleaves viral proteins using the acylation reaction shown in Figure 1. Figure 1  Degradation of viral proteins by acylation of NSP4Computational methods were used to identify the energetic parameters of reactions between NSP4 and synthetic viral proteins.  To ensure that simulated reactions resembled physiological conditions, algorithms were adjusted to account for the enzyme being in an aqueous environment.  The rate-determining step in the acylation reaction was determined by calculating the changes in free energy in both the enzyme and substrate (Figure 2). Figure 2  Free energy profiles of synthetic viral protein cleavageKnowledge of amino acid frequency distribution at an enzymatic active site may facilitate the design of novel active sites and enzyme-specific inhibitors.  Researchers analyzed the composition of amino acids that form the NSP4 active site during protein folding to study the characteristics of residues involved in active site formation (Figure 3). Figure 3  Frequency distribution of amino acid residues at the NSP4 active site Adapted from Stapels DA, Geisbrecht BV, Rooijakkers SH. Neutrophil serine proteases in antibacterial defense. Curr Opin Microbiol. 2015;23:42-8. -Considering that NSPs are proteases, which of the following statements best describes the reaction catalyzed by NSPs?

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. -A researcher hypothesized that cargo delivery is most efficient when the peptide-DNA complex forms prior to DNA interactions with liposomes.  Which additional observation would best support this hypothesis?

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 processes would be directly impaired in von Gierke disease?GlycogenolysisGluconeogenesisPentose phosphate pathway

Passage NDPK is an enzyme in budding yeast that uses ATP to phosphorylate nucleotide diphosphates (NDPs and dNDPs), yielding the triphosphate form of each (NTPs and dNTPs).  dNTPs are necessary for DNA synthesis, yet yeast cells that lack NDPK grow and divide at the same rate as wild-type yeast.  Therefore, triphosphate generation is most likely carried out by one or more other enzymes.The enzyme TMPK converts deoxythymidine monophosphate (dTMP) to the diphosphate form (dTDP) by transferring the γ-phosphate of ATP to dTMP.  Yeast cells that lack TMPK activity cannot synthesize DNA and become arrested during replication.  It was proposed that TMPK may partially compensate for NDPK loss by phosphorylating dTDP as well as dTMP.To better understand the role of TMPK in nucleotide phosphorylation, wild-type TMPK and the inactive E75K variant were purified by affinity chromatography, and the following experiments were performed.Experiment 1After purification, a sample containing 3 μg of protein was added to a reaction mixture containing radiolabeled nucleotide monophosphates (NMPs or dNMPs) and unlabeled ATP.  The reaction proceeded for 30 minutes, and products were separated by thin-layer chromatography (TLC) on a PEI-cellulose plate.  PEI-cellulose separates nucleotides by charge (Figure 1), and migration distance decreases with increasing negative charge.  Spots were visualized by autoradiography, and it was determined that 0.30 nmol of dTMP and 0.15 nmol of uridine monophosphate (UMP) became phosphorylated, respectively. Figure 1  TLC analysis of nucleotide monophosphates after exposure to TMPK and ATP.  (Note: Different nucleotides with the same phosphorylation level migrate different distances.)Experiment 2The experiment was repeated using radiolabeled dTDP as the starting material.  The reaction proceeded in buffer only, in the presence of E75K TMPK, or in the presence of wild-type TMPK.  Products were separated by TLC and visualized (Figure 2). Figure 2  TLC analysis of dTDP phosphorylation with buffer only (Lane 1), E75K TMPK (Lane 2), or wild-type TMPK (Lane 3) Adapted from Chien CY, Chen BR, Chou CK, Sclafani RA, Su JY. The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett. 2009;583(13):2281-6. -Assuming the reaction in Experiment 2 is performed under saturating conditions, which of the following would cause an increase in the rate of phosphorylation?