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- The enzymatic activity of PFK1 is generally measured by set- ting up a coupled enzyme assay system whereby aldolase, triose phos- phate isomerase, and glycerol-3-phosphate dehydrogenase are added to the assay mixture. For the latter enzyme, NADH is added and its change in concentration is readily monitored at 340 nm. Write the chain of reactions catalyzed by these enzymes using structural formulas, label substrates and products, and explain why the coupled en- zyme assay system leads to oxidation of NADH. While the chain of reac- tions is similar to those in glycolysis, there is a critical difference because of the dehydrogenase enzyme. Describe how this enzyme causes the chain of reactions to differ from those in glycolysis.6-Mercaptopurine , after its conversion to the corresponding nucleotide through salvage reactions, is a potent competitive inhibitor of IMP in the pathways for AMP and GMP biosynthesis. It is therefore a clinically useful anticancer agent. The chemotherapeutic effectiveness of 6 mercaptopurine is enhanced when it is administered with allopurinol. Explain the mechanism of this enhancement.Why is de novo biosynthesis of purines markedly elevated in patients with a deficiency in hypoxanthine-guanine phosphoribosyl transferase (HGPRT)? 5-Fluoruracil is converted to methotrexate by a DPD deficiency, leading to increased purine biosynthesis. HGPRT deficiency prevents purine salvage, leading to increased purine biosynthesis to make up for it. HGPRT increases pyrimidine salvage and thereby increases purine biosynthesis as a result. Increased purine biosynthesis is the result of ATP hydrolysis during the production of cyclic AMP.
- Cells defective in thymidylate synthase are able to undergo cell division if methotrexate and thymidine are both provided; however, cells with fully functional thymidylate synthase cannot divide under these same culture conditions. What is the biochemical basis for this observation?The purified OXA-M290 enzyme can now be tested to determine which β-lactamase inhibitor is most effective. This inhibitor could be prescribed in combination with a β-lactam antibiotic to treat the infection caused by the E. coli KGH1 strain. Before testing inhibitors against OXA-M290, the kinetic activity of this enzyme must first be measured. The activity of OXA-M290 is measured using nitrocefin, a chromogenic β-lactam antibiotic. When nitrocefin is hydrolyzed by a β-lactamase, it changes from yellow to red in colour. The nitrocefin hydrolysis product has an extinction coefficient of 20,500 M-1 cm-1 at 486 nm. The hydrolysis of 60 μM nitrocefin by 1 nM OXA-M290 is monitored using a microplate reader. The absorbance of the wells in the plate is measured at 486 nm every 30 seconds. This experiment is carried out with three replicates, generating the following data: Time (min) Absorbance of Replicate 1 Absorbance of Replicate 2 Absorbance of Replicate 3 0.5 0.0984…serine proteases act via a two-step catalytic mechanism. However, as a critical scientist, you may want to see data that supports this claim. One experiment that can provide such evidence is called a 'pre-steady state' kinetics experiment using the chromogenic substrate, N-acetyl- phenylalanine p-nitrophenyl ester. As shown below when this substrate is cleaved, the p-nitrophenylate is a yellow product whose absorbance can be measured in real time using a spectrophotometer. When the chromogenic substrate is rapidly mixed together with a limiting amount of protease enzyme, the accumulation of product is measured right away on fast (millisecond) timescales. In this experiment, one observes two distinct phases of the reaction as shown in the plot below. Based upon what you know about the mechanism of serine proteases, provide a mechanistic interpretation for the observation of these two phases of the reaction. + H₂O N-Acetyl-L-phenylalanine p-nitrophenyl ester p-Nitrophenolate Absorbance…
- B-lactamase is an enzyme found in many antibiotic-resistant bacteria that hydrolyzes and inactivates antibiotics like penicillin and cephalosporin. The amount of antibiotic hydrolyzed in 1 minute in a 10-ml solution containing purified ß-lactamase was measured as a function of antibiotic concentration. The kinetics of hydrolysis was performed for two antibiotic substrates (A and B). Assume that the concentration of ß-lactamase was kept constant during the assay. 12 Initial Velocity (nanomole/min) 10 8 2 0 10 20 | 30 [Antibiotic] (µm) 40 50 Antibiotic A Antibiotic B a) Based on the enzyme description, what type of enzyme is ß-lactamase? Lyase Isomerase Ligase Hydrolase Oxidoreductase Transferase b) Based on your answer in (a), what other reactant, in addition to the antibiotic substrate, needs to be in the active site of ß-lactamase for the hydrolysis reaction to proceed? c) From the reaction curves above, what is the approximate value of Vmax for the enzyme reaction? (Do not forget the…Dihydrofolate reductase (DHFR) is an enzyme that reduces 7,8-dihydrofolate (DHF) to 5,6,7,8- tetrahydrofolate, a step in the biosynthesis of thymidine. Methotrexate (MTX) a structural analogue of dihydrofolate, inhibits this enzyme. MTX has been used in cancer therapies because the inhibition of DHFR restricts thymidine production required for cell division. In the absence of thymidine the cancerous cells cannot multiply. Using the following data of DHFR activity in the presence of MTX, i) Determine the inhibition mechanism of MTX and the maximum reaction rate. If Km for DHFR is 0.100 uM, what is K; ? ii) [DHF](MM) Rate w/200 nM Rate w/50 nM MTX (mMs-¹) Rate w/100 nM MTX MTX (mMs ¹) (mMs ¹) 3 7.38 5.56 3.72 6 8.84 7.38 5.55 9 9.46 8.29 6.65 12 9.79 8.84 7.38AMP- PNP is a non-hydrolyzable ATP analog that cannot be metabolized by cells. Taurocholate is a bile acid that helps emulsify fats. When taurocholate is added to hepatocyte cell culture, it accumulates in those cells. The graph below shows the rate of cellular accumulation of the drug taurocholate in the presence of either ATP, ATP, or AMP-PNP. Based on this date, describe the mechanism by which taurocholate enters the cell. Justify the answer.
- The mechanism involved in the reaction catalyzed by phosphoglyceromutase is known to involve a phosphorylatedenzyme intermediate. If 3-phosphoglycerate is radioactively labeledwith 32P, the product of the reaction, 2-phosphoglycerate, does nothave any radioactive label. Design a mechanism to explain these facts.TPCK and TLCK are irreversible inhibitors of serine proteases. One ofthese inhibits trypsin and the other chymotrypsin. Which is which? Explainyour reasoning. Suggest the effects of each of the following mutations on the physiologicalrole of chymotrypsinogen:(a) R15S(b) C1S(c) T147SPhosphoglycerate kinase shows induced fit resulting from the binding of both substrates to form the closed catalytic conformation. Maintainence of the closed conformation depends on the formation of a salt bridge between R62 and D200. What would be the effect of the mutation of D200 to the unusual amino acid shown below? Explain.