Biochemistry-Pt.I Mid-term exam Block 3, 2008 Name Answer any 10 of the following 13 questions (all are worth 8 pts). Be sure to include molecular details in your answers that support the cause and effect relationships that exist between the chemical mechanisms and physiological results. Part II of this exam is a 20 point case study that is to be handed in no later than 9am tomorrow morning. You may pick this up as you leave the exam today. Please, do not consult with other people on this part of the exam although you may use other resources if you find it necessary. 1. Outline the metabolic relationships among the liver, brain, muscle, and adipose tissue in the fasting state (assume the person is at rest). Be sure to identify the primary fuel source used by each organism and where it is originating. 2. Glucagon and epinephrine elicit similar metabolic responses but differ in the tissue they affect; myocytes lack the glucagon receptor and thus do not respond to this hormone. Rationalize the lack of glucagon receptors with respect to the metabolic relationships between, and functions of, the liver and muscle tissue. Glucagon signals/triggers mobilization of glycogen fuel reserves. Liver glycogen is continuously made and degraded during the course of a day and serves as a glucose reserve used to help maintain homeostasis of blood glucose levels. Muscle glycogen, on the other hand, is a fuel reserve only for the muscles. This glycogen is reserved until physical activity demands large amounts of energy for muscle movement. If muscles responded to glucagon, glycogen would be depleted as a function of diet and may not be on reserve when needed (like when chasing food when you are hungry). Because muscles are the only users of their glycogen stores, it makes sense to have glycogenolysis respond to a fright or flight hormone like epinephrine, and not to a fed/fasting signal like glucagon. Exam_midterm_Ansrs 1 of 7
3. Several years ago, the Atkin s diet gained popularity as a weight loss program. A very crude description of this diet plan is that it advocates reducing carbohydrates and increasing protein consumption in the diet. The figure below illustrates changes in blood chemistry after meals emphasizing either carbohydrates or proteins. How would the Atkin s approach to weight loss work in other words, what is the mechanism behind this weight loss strategy? For carbohydrate meal: There is a big change in the relative amounts of glucagon and insulin as a result of glucose consumption. The insulin increase and glucagon decrease signal the body to store glucose as glycogen or process it via glycolysis into acetyl-coa and then use that to produce fatty acids/tags that can be stored in the adipocytes. For the protein meal: The changes in insulin levels following the meal are dramatically reduced, thus providing less of a signal requesting fuel storage. Furthermore, glucagon level actually increases in this situation signaling the body to mobilize fuel reserves. As a result, the balance of fuel storage/mobilization is redistributed, presumably to favor mobilization. While it is undoubtedly much more complicated than that, it at least provides some sense of entitlement for the Atkin s people to claim that.eating the right foods while limiting refined carbohydrates in one s diet change[s] a person s body from a carb-burning to a fat-burning machine. Despite his diet program, Dr. Atkins eventually died anyway. 4. GLUT2 is a pancreatic glucose transporter serving as a glucose sensor that ultimately controls insulin release. a) How would a mutation in the GLUT2 allele that increases the protein s K d for glucose, affect the control mechanism in the islet cell? Be sure to include enough biochemical detail to describe the cause and effect relationships of the release mechanism. Increasing K d reduces the affinity of the transporter for glucose. Therefore, the transporter will import less glucose into the islet cell. Less glucose in the cytosol reduces flux through glycolysis and as a result reduces ATP production. The K + transporter that is part of the triggering mechanism for insulin release is sensitive to ATP levels; when ATP levels are reduced, the tranporter is able to maintain the membrane potential. It is the loss of membrane potential that normally triggers insulin release due to an influx of Ca 2+ via a voltage-gated transporter. The fact that the membrane potential is maintained, Ca 2+ influx is prevented and thus insulin is not released. b) Predict if an individual with this mutation would tend to be hypo- or hyperglycemic? Obviously, if there is little insulin being released, blood glucose will remain high since glucose uptake in other tissues has not been turned on. Exam_midterm_Ansrs 2 of 7
5. We examined the opposing regulatory cascades of glucagon and insulin controlling glycogen synthesis and degradation in the liver. A similar process is occurring in the adipose tissue to control the futile cycling of triacylglycerol (TAG) synthesis and degradation. a) Read the short passage from the text (on a separate handout) describing lipolysis regulation via glucagon and construct a map showing the causal relationships between low blood glucose and lipolysis in adipocytes. b) Propose a counter-regulatory cascade that decreases lipolysis and is caused by high blood glucose levels. You should be aware that insulin up-regulates Insulin-Sensitive Protein Kinase which in turn regulates Phosphoprotein Phosphatase 1 (PP1). (you will need to figure out which way PP1 is regulated) 6. In the case of lipid metabolism, about 70% of all free fatty acids released by lipolysis ends up being remade into TAGs, either in the liver or in adipocytes, even in a fasting state. Justify the existence of this seemingly futile cycle. Cycling of opposing pathways allow for a highly adjustable response to a regulatory signal. These cycles can be adjusted over such a wide range because the opposing pathways are often controlled independently of one another and can be used to control flux through a pathway. In this case, the cycling provides for a constant supply of free fatty acids for muscle metabolism since the resynthesis occurs in both the liver and the adipocytes. Exam_midterm_Ansrs 3 of 7
7. Develop a semi-quantitative model describing the change of flux through the fatty acid/tag cycle when the body switches from fed to fasting state. Assume that, (1) at their lowest active state, TAG synthesis has 5 units of activity and TAG degradation has 10 units of activity. (2) in the fed state, insulin stimulates TAG synthesis by 10x (3) in the fasting state glucagon stimulates TAG degradation by 50x What relative change in flux for TAG metabolism could you expect given these parameters? What regulatory concept does this model illustrate about controlling pathway flux? TAG synthesis TAG degradation Flux (to syn) Fed state 50 10 +40 Fasting state 5 500-495 The differential regulation allows for a large change in flux relative to TAG synthesis. If the TAG synthesis and degradation simply relied on an equilibrium between these two process (i.e. if you increase synthesis by the same amount you decrease degradation), you could only expect a change or +/- 40 units for switching between forward (synthesis) and reverse (degradation) direction. However, because these processes are oppositely regulated and are affected by different degrees, the combined effect of changes to TAG synthesis and degradation is about 100x greater (500 vs 40). 8. The T R transition model is general tool to describe the cooperativity in other enzymes, not just Hb. Above is a schematic diagram of the T (Fig. a) and R (Fig. b) states of PFK. Use this information to predict how the following mutations would affect the T R equilibrium and how the mutations would be expected to affect enzyme activity. a) Glu161 Gln (glutamine; see me if you need reminded of the functional group) This mutation causes a loss of stabilizing interactions across the subunit boundary, destabilizing the T state. This pushes the equilibrium toward the R state favoring binding of the substrate. This effect would be expected to increase the enzyme activity. b) Arg72 Lys (lysine) Again, this mutation would destabilize the T state by removing inter-subunit interactions, thus pushing the equilibrium toward the R state. However, because this residue also provides binding interactions for substrate, replacing arginine would like limit substrate binding and thus have the effect of decreasing enzyme activity. Exam_midterm_Ansrs 4 of 7
9. Compare and contrast the general structures of myoglobin and hemoglobin and explain why each is particularly well suited to their physiological function. Be sure to provide sufficient biochemical detail describing molecular mechanisms and graphs illustrating binding differences. Myoglobin: 1. single polypeptide exhibiting a high affinity for O 2 binding. 2. binding curve for O 2 is hyperbolic indicating no cooperativity (and we would not expect any due to the lack of subunits) 3. The physiological function is to increase O 2 solubility in the tissues generating a steeper concentration gradient to the mitochondria and increase the efficiency of O 2 delivery. Hemoglobin: 1. tetrameric protein exhibiting a variable affinity for O 2. 2. binding curve for O 2 is sigmoidal indicating a cooperative binding effect. We see that there is more binding than we might expect for a hyperbolic curve at higher O 2 pressures, and less binding than expected at lower O 2 pressures. 3. The cooperative binding properties are due to conformational changes within the protein. Binding of O 2 to the heme changes the relative position of an α-helix that is coordinating the heme iron. This shifting is propagated to adjacent subunits by altering the intermolecular forces stabilizing the T or deoxy form. Destabilizing the T state moves the protein more toward the R state which has a higher affinity for O 2 thus encouraging additional binding of O 2. 4. Physiologically, cooperative binding allows for variations in binding affinity allowing Hb to off-load O 2 where po 2 values are low (in the tissues) but load up on O 2 when po 2 values are high (as in the lungs). Thus, Hb is particularly well suited for O 2 transport. 10. The Bar-Headed Goose migrates over Mt. Everest, and sequencing of the Hb coding regions has revealed a proline alanine mutation of residue 119 in the alpha subunit. This mutation eliminates a Van der Waals contact with residue 55 of the β subunit and destabilizes the T state of Hb. What physiological function does this serve? What problem might it cause for the goose and how could it be rectified? Loss of the VanderWaals interaction shifts the Hb equilibria to favor the R state allowing the protein to bind more O 2. This is relevant for functioning at higher altitudes where po 2 values are significantly lower than at sea level and would assist the goose in scavenging more O 2. However, this also poses a problem in deoxygenating Hb within the tissues since it would have a higher binding affinity. One way around this is if the goose possesses a higher concentration of some allosteric effector like 2,3- bisphosphoglycerate (BPG). BPG helps to stabilize the T state favoring deoxygenation of Hb. Thus it may be that while the Hb mutation may help increase O 2 binding, there are undoubtedly other physiological adaptations that fine tune Hb function in this environment. Exam_midterm_Ansrs 5 of 7
11. Penicillin resistance in bacteria is caused by β-lactamase, an enzyme that cleaves the core lactam structure of penicillin antibiotics. Ampicillin and oxacillin are two β-lactam antibiotics degraded by β -lactamase. Use the kinetic parameters below to identify which of these antibiotics is more efficiently degraded by the enzyme. Be sure to provide an explanation for your decision. k cat (sec -1 ) k m (M) k cat /k m ampicillin 4 x 10 2 1.4 x 10-4 2.9 x 10 6 oxacillin 9 x 10 3 2.3 x 10-3 3.9 x 10 6 It isn t possible to evaluate efficiency from either kcat or km alone. In this case ampicillin is a better substrate but processed more slowly, while oxacillin is processed fast but is a poorer substrate. Only by using kcat/km will you normalize each of these properties for a fair comparison. From this ratio, we find that oxacillin is more efficiently degraded by the lactamase; apparently the fact that it is a better substrate outweighs the slower turnover. 12. The data set below describes the reaction velocity for an enzyme both in the presence (treated column) and the absence (untreated column) of an inhibitor. S conc. (um) Velocity-untreated (umol/min) Velocity-treated (umol/min) 10 49 57 20 96 118 80 349 223 250 545 360 500 655 476 1000 690 598 2000 699 650 a) Estimate the k m and V max for this enzyme. Explain in one sentence each, how you found these parameters. V max ~700 umol/min the velocity data tend towards this value and is the asymptote of the hyperbolic curve. K m is [S] when V is ½ V max ; therefore ~ 80 um b) Classify the type of inhibitor being used and explain what information you used to classify it. This must be a competitive inhibitor since the V max remains unchanged while the k m increases to ~ 250 um Exam_midterm_Ansrs 6 of 7
13. Aldolase catalyzes the step in glycolysis where the hexose sugar is broken into 2 trioses (DHAP and GAP, if you really have to know). a) Below is a schematic diagram describing the catalytic mechanism. Identify three catalytic strategies used in the aldolase mechanism that allows this reaction to proceed. transition state stabilization: enzyme binds the substrate to make it look more like the transition state, thus lowering activation energy covalent catalysis: substrate is bonded to enzyme via imine linkage proximity/orientation effects: enzyme arranges orbitals of the substrate and catalytic residues to overlap for the reaction to occur Acid/base catalysis: basic residue removes proton from substrate b) There are actually two classes of aldolases, one of which is a metalloprotein and employs a Zn +2 ion to catalyze the reaction. These aldolases lack the lysine residue used to form the Schiff base with the substrate. In what capacity could the Zn +2 ion make up for the loss of this lysine residue to assist in the catalysis? Exam_midterm_Ansrs 7 of 7