Exam 2: Biochemistry

Passage Upon entering muscle cells, glucose is immediately phosphorylated by hexokinase to produce glucose 6-phosphate (G6P).  This phosphorylation event prevents glucose from exiting the cell.  G6P may then enter one of three metabolic pathways, depending on the needs of the cell (Figure 1). Figure 1  Three potential fates of G6P, determined by the needs of the cellUnder low ATP conditions, G6P enters glycolysis, where it is converted to two pyruvate molecules.  The energy released in this process produces a net total of two ATP molecules per glucose molecule consumed.  The resulting pyruvate molecules can then enter the mitochondria, where they participate in additional metabolic reactions that produce more ATP.When the cell experiences oxidative stress, G6P enters the pentose phosphate pathway (PPP) and produces NADPH, a reducing agent.  During this process, G6P loses one carbon atom in the form of CO₂ and is converted to ribose 5-phosphate (R5P), which is required for nucleotide synthesis.  In addition to low NADPH conditions, G6P may also enter the PPP during replication, when nucleotides are needed for DNA synthesis.  If nucleotides are not needed but energy is, R5P can be converted back to glycolytic intermediates and reenter glycolysis.  Each glucose molecule that is converted to R5P prior to glycolysis yields an average of 1.67 ATP molecules.When all the cell's metabolic requirements are met, additional G6P can enter glycogenesis and produce glycogen, which stores glucose for later use; this process consumes ATP.  When signal molecules bind G protein-coupled receptors on muscle cells, glycogenolysis is activated to release individual monomers from glycogen, allowing them to enter the appropriate pathway. -Based on information in the passage, if glucose enters the pentose phosphate pathway prior to glycolysis, what is the energy loss compared to glucose that enters glycolysis directly?  (Note: The free energy of ATP hydrolysis is −30 kJ/mol.)

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. -5-AVAB most likely alters fatty acid metabolism in cardiomyocytes through which mechanism?

A protein denatures as ionic interactions are disrupted by deprotonation of positively charged side chains.  This is most likely the result of:

Passage Although the structure of the cell membrane has been investigated repeatedly, few studies examine the contribution of its lipid components.  Lipid composition influences the fluidity and permeability of cell membranes in the human body.  Chronic diseases such as atherosclerosis and psoriasis arise from alterations to the lipid composition.Atherosclerosis is caused by the accumulation of cholesterol in the endothelial cells lining the arterial wall.  Cholesterol carried by low-density lipoprotein (LDL) is deposited into cell membranes, where it forms cholesterol crystalline domains (CCDs).  In damaged cells, these CCDs increase membrane permeability and contribute to the formation of cholesterol plaques that can potentially block the artery itself.Omega-3 fatty acids (O3FAs) derived from marine animals may be used to slow the development of atherosclerosis.  Formulations of O3FA contain varying concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Figure 1).  Present research suggests that formulations of O3FA should contain EPA exclusively because it prevents cholesterol from organizing into CCDs.  Nonetheless, atherosclerosis is generally treated by administering drugs referred to as statins, which decrease the free cholesterol in blood plasma by inhibiting the production of a cholesterol precursor.  Statin therapy combined with O3FAs may be used to reduce atherosclerosis in patients with persistent hypertriglyceridemia (high triglyceride plasma levels). Figure 1  Organization of membranes in endothelial and epidermal cellsWater loss from the uppermost layer of the epidermis, the stratum corneum (SC), is prevented by specialized lipids called ceramides (cer).  Psoriasis is a skin disease in which the permeability of the SC is compromised due to a significant decrease in the level of ceramides.  The scarcity of ceramides impedes the formation of a lipid matrix called the long periodicity phase (LPP) in the cell membrane.  Increasing the concentration of ceramides such as esterified omega-hydroxyacyl-sphingosine (cer-EOS) has been shown to reduce the permeability of SC cell membranes and may be used to treat psoriasis scabs (Figure 1). Adapted from Mason RP, Jacob RF, Shrivastava S, Sherratt SC, Chattopadhyay A. Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes. Biochim Biophys Acta. 2016;1858(12):3131-3140. -Phospholipids in the cell membrane could be separated experimentally according to their differences in:charge.mass.solubility.

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. -The active site of wild-type ALDH2 most likely interacts with which of the following amino acid residues on the antibodies shown in Figure 1?

A protein folds spontaneously in phosphate-buffered saline (PBS).  Guanidinium chloride is gradually titrated into the PBS-buffered protein solution, causing the protein to denature.  During the titration, the ΔG° of protein folding is:

A single-stranded DNA oligonucleotide composed of which of the following would move most slowly down an alkaline agarose gel during electrophoresis?

Passage Leukocyte common antigen (LCA) enzymes play several important developmental roles in both vertebrates and invertebrates.  They regulate numerous cellular processes by catalyzing Reaction 1 in target proteins at specific developmental stages.  Consequently, LCA enzyme expression and activity are tightly controlled in various cell types. Reaction 1The Src protein is critical for cell differentiation and motility, and is activated by LCA enzyme activity.  Two LCA enzymes, known as LAR and CD45, were purified and incubated with the phosphopeptide src527 (Figure 1), derived from Src. Figure 1  Structure of the src527 peptide used as a substrate in LAR and CD45 assaysThe rate of Reaction 1 was measured at various concentrations of src527 in the presence of either LAR or CD45, and kinetic parameters were determined (Table 1).Table 1  Kinetic Parameters of LAR and CD45 Compound 1 was synthesized and tested for its effectiveness as an inhibitor of LCA enzymes. The study in Table 1 was repeated in the presence of Compound 1, which was found to significantly reduce the activities of both LAR and CD45 against the src527 peptide.  Compound 1 was subsequently used in assays to examine the in vivo function of LCA enzymes. Adapted from Cho H, Krishnaraj R, Itoh M, et al. Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors. Protein Sci. 1993;2(6):977-84. -The information in the passage suggests that the effects of LCA activity could be counteracted by which type of enzymatic activity?

Passage Human gamma-glutamyl transpeptidase (hGGT) is an enzyme involved in cysteine homeostasis, and its overexpression has been linked to asthma, cancer, and other diseases.  It catalyzes the addition of water across a bond to cleave extracellular glutathione (GSH) into glutamate and a dipeptide composed of cysteine and glycine.  It is believed to function by the mechanism shown in Figure 1. Figure 1  Proposed mechanism of hGGT-mediated GSH cleavageResearchers interested in investigating hGGT inhibitors as a treatment for pathologies related to excess hGGT activity performed the following experiments, each containing the same concentration of hGGT.Experiment 1hGGT was incubated with various concentrations of GSH in the presence or absence of 250-μM candidate compound acivicin or Compound 1.  However, both candidates were found to be unsuitable as acivicin showed significant neurotoxicity and Compound 1 acted as an activator instead of an inhibitor. Figure 2  Lineweaver-Burk plot of hGGT activity with respect to GSH in the presence or absence of 250-μM candidate inhibitorsExperiment 2Researchers hypothesized that slight variations in the structure of Compound 1 could result in hGGT inhibition and tested several derivatives, shown in Table 1.Table 1  Kinetic Parameters of hGGT in the Presence or Absence of Several Candidate Inhibitors at 250 μM Experiment 3To better understand the physiological conditions under which hGGT operates, researchers exposed a cultured cell line expressing hGGT on the cell surface to a solution that mimicked the composition of extracellular fluid, including levels of GSH.  They found that in this setup, uninhibited hGGT results in a reaction rate that is approximately ½V_max. Adapted from Wickham S, Regan N, West MB, et al. Inhibition of human ?-glutamyl transpeptidase: development of more potent, physiologically relevant, uncompetitive inhibitors. Biochem J. 2013;450(3):547-57. -Based on Figure 2, Compound 1 activates hGGT in which of the following ways?

The reaction between UTP and glucose-1-phosphate (G1P) to form UDP-glucose in glycogenesis is shown below: Which phosphates from the reactants are present in UDP-glucose?

Passage Although the structure of the cell membrane has been investigated repeatedly, few studies examine the contribution of its lipid components.  Lipid composition influences the fluidity and permeability of cell membranes in the human body.  Chronic diseases such as atherosclerosis and psoriasis arise from alterations to the lipid composition.Atherosclerosis is caused by the accumulation of cholesterol in the endothelial cells lining the arterial wall.  Cholesterol carried by low-density lipoprotein (LDL) is deposited into cell membranes, where it forms cholesterol crystalline domains (CCDs).  In damaged cells, these CCDs increase membrane permeability and contribute to the formation of cholesterol plaques that can potentially block the artery itself.Omega-3 fatty acids (O3FAs) derived from marine animals may be used to slow the development of atherosclerosis.  Formulations of O3FA contain varying concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Figure 1).  Present research suggests that formulations of O3FA should contain EPA exclusively because it prevents cholesterol from organizing into CCDs.  Nonetheless, atherosclerosis is generally treated by administering drugs referred to as statins, which decrease the free cholesterol in blood plasma by inhibiting the production of a cholesterol precursor.  Statin therapy combined with O3FAs may be used to reduce atherosclerosis in patients with persistent hypertriglyceridemia (high triglyceride plasma levels). Figure 1  Organization of membranes in endothelial and epidermal cellsWater loss from the uppermost layer of the epidermis, the stratum corneum (SC), is prevented by specialized lipids called ceramides (cer).  Psoriasis is a skin disease in which the permeability of the SC is compromised due to a significant decrease in the level of ceramides.  The scarcity of ceramides impedes the formation of a lipid matrix called the long periodicity phase (LPP) in the cell membrane.  Increasing the concentration of ceramides such as esterified omega-hydroxyacyl-sphingosine (cer-EOS) has been shown to reduce the permeability of SC cell membranes and may be used to treat psoriasis scabs (Figure 1). Adapted from Mason RP, Jacob RF, Shrivastava S, Sherratt SC, Chattopadhyay A. Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes. Biochim Biophys Acta. 2016;1858(12):3131-3140. -Individuals undergoing statin therapy would most likely exhibit:

Passage Leukocyte common antigen (LCA) enzymes play several important developmental roles in both vertebrates and invertebrates.  They regulate numerous cellular processes by catalyzing Reaction 1 in target proteins at specific developmental stages.  Consequently, LCA enzyme expression and activity are tightly controlled in various cell types. Reaction 1The Src protein is critical for cell differentiation and motility, and is activated by LCA enzyme activity.  Two LCA enzymes, known as LAR and CD45, were purified and incubated with the phosphopeptide src527 (Figure 1), derived from Src. Figure 1  Structure of the src527 peptide used as a substrate in LAR and CD45 assaysThe rate of Reaction 1 was measured at various concentrations of src527 in the presence of either LAR or CD45, and kinetic parameters were determined (Table 1).Table 1  Kinetic Parameters of LAR and CD45 Compound 1 was synthesized and tested for its effectiveness as an inhibitor of LCA enzymes. The study in Table 1 was repeated in the presence of Compound 1, which was found to significantly reduce the activities of both LAR and CD45 against the src527 peptide.  Compound 1 was subsequently used in assays to examine the in vivo function of LCA enzymes. Adapted from Cho H, Krishnaraj R, Itoh M, et al. Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors. Protein Sci. 1993;2(6):977-84. -Which of the following calculations was used to determine the catalytic efficiencies in Table 1?

Passage Many lysosomal proteins are transported to the lysosome by the cation-independent mannose 6-phosphate receptor (CI-MPR).  CI-MPR is a transmembrane protein found in the Golgi apparatus and vesicles.  It contains 15 globular domains folded similarly on the luminal side of the membrane, each of which has up to 3 glycosylation sites and 6−12 cysteine residues that aid in folding.The ability of CI-MPR to recognize multiple different ligands was studied by fluorescent microarray analysis.  Microarray analysis was performed by cross-linking candidate ligands such as plasminogen, insulin-like growth factor 2 (IGF-II), and mannose 6-phosphate (M6P), to glass slides.  The slides were then incubated with full-length CI-MPR or recombinantly expressed CI-MPR domains, followed by incubation with an anti−CI-MPR antibody.  A fluorescently labeled antibody that binds the first antibody was then added, and the relative fluorescence levels of each candidate ligand were compared.  Higher fluorescence levels correspond to greater binding.Experiment 1Microarray analysis was carried out on slides containing varying levels of candidate ligands, and binding affinities of each CI-MPR domain for its primary ligand were estimated.  Table 1 shows the results.Experiment 2Domains 3, 5, and 9 bind carbohydrates.  Amino acids in these domains were mutated to determine which are required for binding.  Based on this experiment, scientists concluded that Arg, Glu, and Tyr are required for carbohydrate binding by domains 3, 5, and 9 of CI-MPR.Table 1 Adapted from Bohnsack RN, Song X, Olson LJ, et al. Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites. J Biol Chem. 2009;284(50):35215-26. -Which class of amino acids is LEAST likely to be found on the surface of CI-MPR globular domains?

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. -As NSP4 folds, the water molecules surrounding it become:

Escherichia coli bacteria containing only ¹⁵N-labeled DNA were grown in media containing only ¹⁴N nucleotides.  What percentage of double helices would be composed of one ¹⁵N strand and one ¹⁴N strand after three generations?

Passage Dystroglycan (Dg) is a transmembrane protein that mediates interactions between the cytoskeleton and the extracellular matrix (ECM).  It binds the ECM protein laminin in an interaction that is required for intercellular communication and muscle membrane structural stability.  The Dg-laminin interaction is facilitated by a long carbohydrate chain called an O-mannosyl glycan, which is covalently linked to Dg (Figure 1). Figure 1  Schematic of interaction between Dg and laminin, mediated by the Dg-linked O-mannosyl glycanThe O-mannosyl glycan is added to Dg by a series of enzymes called glycosyltransferases.  The first enzyme in this series, protein O-mannosyltransferase (POMT), attaches D-mannose to the hydroxyl groups of serine and threonine residues in an α-linkage through the anomeric carbon.  The next sugar in the chain then forms a β-1,4 linkage to the mannose residue, followed by several more steps to produce the full glycan.  Genetic inactivation of any of the enzymes in the pathway results in an incomplete glycan and failure to bind laminin, and is the underlying cause of several severe forms of muscular dystrophy known as dystroglycanopathies.Researchers investigating dystroglycanopathies recently discovered the functions of two enzymes in the pathway:  protein O-mannose kinase (POMK) and fukutin.  POMK phosphorylates Dg-linked mannose at carbon 6, and fukutin adds the sugar ribitol 5-phosphate to the glycan chain.  To assess the roles of POMK and fukutin relative to each other, scientists conducted experiments in which lysates from muscle cells deficient in either POMK or fukutin were incubated with ³²P-labeled ATP or ³H-labeled activated ribitol 5-phosphate.  Combined lysates containing both fukutin and POMK were also tested.  Dg was then purified and detected by autoradiography and western blot using an antibody against Dg, as shown in Figure 2. Figure 2  Autoradiograms and western blots of Dg purified from lysates incubated with ³²P-labeled ATP or ³H-labeled ribitol 5-phosphate Adapted from Kanagawa M, Kobayashi K, Tajiri M, et al. Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy. Cell Rep. 2016;14(9):2209-23. -Which conclusion about the functions of POMK and fukutin is supported by the autoradiogram in Figure 2?

Passage The tumor suppressor p53 is a homotetrameric protein with an N-terminal transactivation domain (TAD), a proline-rich domain, a DNA-binding domain (DBD), a tetramerization domain (TD), and a C-terminal regulatory domain (REG) on each subunit (Figure 1). Figure 1  Organization of the p53 domains (1-393 amino acids)Human double minute 2 (Hdm2) is a lysine-specific ubiquitin protein ligase that can change the half-life of some proteins, including p53, by signaling for their proteolytic cleavage.  Under normal circumstances, Hdm2 binds p53 at the AD1 region within the TAD and attaches ubiquitin to a lysine residue.  However, errors in DNA replication induce the phosphorylation of residue T18 on p53, which inhibits p53's association with Hdm2.  The overexpression of Hdm2 has been correlated with the downregulation of tumor suppressor p53 in certain types of cancers.  Changes in the NMR chemical shift (ppm) of amino acids in the AD1 region shown in Figure 2 provide evidence for the formation of a p53-Hdm2 complex. Figure 2  ¹H NMR spectrum of the AD1 region in ¹⁵N-labeled p53 during G₀ and G₂ phases of the cell cycleHdm2 also binds to Nedd8 ultimate buster 1 (NUB1), which enhances signaling for the degradation of Nedd8 proteins.  Preliminary data suggest that Hdm2 interacts differently with wild-type NUB1 (WTNUB1) than with mutant K159RNUB1.  To assess the effect of Hdm2 on NUB1 and p53, the half-lives of p53 and Nedd8 protein variants were compared in cells that overexpress Hdm2 (Figure 3). Figure 3  Half-lives of p53, phosphorylated p53 (p53-PO₄), and Nedd-8 proteins in cells with wild-type or mutant NUB1 over time Adapted from Ferreon JC, Lee CW, Arai M, Martinez-yamout MA, Dyson HJ, Wright PE. Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2. Proc Natl Acad Sci USA. 2009;106(16):6591-6. -The data in Figure 3 support which of the following conclusions?

Passage Human gamma-glutamyl transpeptidase (hGGT) is an enzyme involved in cysteine homeostasis, and its overexpression has been linked to asthma, cancer, and other diseases.  It catalyzes the addition of water across a bond to cleave extracellular glutathione (GSH) into glutamate and a dipeptide composed of cysteine and glycine.  It is believed to function by the mechanism shown in Figure 1. Figure 1  Proposed mechanism of hGGT-mediated GSH cleavageResearchers interested in investigating hGGT inhibitors as a treatment for pathologies related to excess hGGT activity performed the following experiments, each containing the same concentration of hGGT.Experiment 1hGGT was incubated with various concentrations of GSH in the presence or absence of 250-μM candidate compound acivicin or Compound 1.  However, both candidates were found to be unsuitable as acivicin showed significant neurotoxicity and Compound 1 acted as an activator instead of an inhibitor. Figure 2  Lineweaver-Burk plot of hGGT activity with respect to GSH in the presence or absence of 250-μM candidate inhibitorsExperiment 2Researchers hypothesized that slight variations in the structure of Compound 1 could result in hGGT inhibition and tested several derivatives, shown in Table 1.Table 1  Kinetic Parameters of hGGT in the Presence or Absence of Several Candidate Inhibitors at 250 μM Experiment 3To better understand the physiological conditions under which hGGT operates, researchers exposed a cultured cell line expressing hGGT on the cell surface to a solution that mimicked the composition of extracellular fluid, including levels of GSH.  They found that in this setup, uninhibited hGGT results in a reaction rate that is approximately ½V_max. Adapted from Wickham S, Regan N, West MB, et al. Inhibition of human ?-glutamyl transpeptidase: development of more potent, physiologically relevant, uncompetitive inhibitors. Biochem J. 2013;450(3):547-57. -Mutating residue 381 to which of the following amino acids would most likely result in the smallest change in hGGT activity?

Passage The ABO blood groups are determined by the polysaccharide antigens linked to sphingolipids on red blood cell membranes.  The polysaccharide known as the H-antigen (O-type blood) consists of a polysaccharide (R-group) glycosidically linked to D-galactose, which is connected to L-fucose in an α-1,2 linkage.  Type A and type B blood express the A- and B-antigens, respectively (Figure 1).  These antigens are modified forms of the H-antigen, in which D-galactose (B-antigen) or a derivative of D-galactose called GalNAc (A-antigen) is added in an α-1,3 linkage to the D-galactose moiety of the H-antigen. Figure 1  Structures of the H-, A-, and B-antigens.  The R-group represents additional carbohydrates.The addition of GalNAc or galactose to the H-antigen is catalyzed by an enzyme encoded by the ABO gene.  One allele of the gene encodes GalNAcyl transferase and produces the A-antigen, while another allele encodes galactosyl-transferase and yields the B-antigen (Figure 1).  The two alleles differ at seven nucleotide positions, summarized in Table 1.Table 1  Nucleotide Positions Where the Alleles Encoding GalNAcyl Transferase and Galactosyl Transferase Differ Individuals with both alleles express both antigens and have type AB blood.  Those with type O blood have a third allele that encodes an inactive form of the enzyme.  A study of blood type abundance was conducted, and the number of participants with each type is shown in Table 2.  Table 3 shows several fatty-acyl chains found in the sphingolipids to which the antigens were attached.Table 2  Number of Participants That Express Each Blood Type in a Study of 10,000 Individuals Table 3  Types of Sphingolipids to Which Polysaccharide Antigens Were Bound, as Determined by Mass Spectrometry (Note:  For fatty acid chains, the numbers on the left and right of the colon indicate number of carbons and number of C=C double bonds, respectively) -Approximately what percentage of the population measured in Table 2 expresses GalNAcyl transferase?

Passage The ABO blood groups are determined by the polysaccharide antigens linked to sphingolipids on red blood cell membranes.  The polysaccharide known as the H-antigen (O-type blood) consists of a polysaccharide (R-group) glycosidically linked to D-galactose, which is connected to L-fucose in an α-1,2 linkage.  Type A and type B blood express the A- and B-antigens, respectively (Figure 1).  These antigens are modified forms of the H-antigen, in which D-galactose (B-antigen) or a derivative of D-galactose called GalNAc (A-antigen) is added in an α-1,3 linkage to the D-galactose moiety of the H-antigen. Figure 1  Structures of the H-, A-, and B-antigens.  The R-group represents additional carbohydrates.The addition of GalNAc or galactose to the H-antigen is catalyzed by an enzyme encoded by the ABO gene.  One allele of the gene encodes GalNAcyl transferase and produces the A-antigen, while another allele encodes galactosyl-transferase and yields the B-antigen (Figure 1).  The two alleles differ at seven nucleotide positions, summarized in Table 1.Table 1  Nucleotide Positions Where the Alleles Encoding GalNAcyl Transferase and Galactosyl Transferase Differ Individuals with both alleles express both antigens and have type AB blood.  Those with type O blood have a third allele that encodes an inactive form of the enzyme.  A study of blood type abundance was conducted, and the number of participants with each type is shown in Table 2.  Table 3 shows several fatty-acyl chains found in the sphingolipids to which the antigens were attached.Table 2  Number of Participants That Express Each Blood Type in a Study of 10,000 Individuals Table 3  Types of Sphingolipids to Which Polysaccharide Antigens Were Bound, as Determined by Mass Spectrometry (Note:  For fatty acid chains, the numbers on the left and right of the colon indicate number of carbons and number of C=C double bonds, respectively) -Which of the following statements accurately describes the sphingolipids in Table 3?They provide structure to biological membranesThey are primarily used for energy storageThey can be hydrolyzed to produce two fatty acids