The enzyme glucose oxidase isolated from the mold Penicillium notatum catalyzes the oxidation of B-D-glucose to D-glucono-8-lactone. The enzyme is highly specific for the B anomer of glucose and does not affect the a anomer. In spite of this specificity, the reaction catalyzed by glucose oxidase is commonly used in a clinical assay for total blood glucose -that is, for solutions consisting of a mixture of B- and a-D-glucose, as well as other sugars present in blood. The oxidation proceeds in the presence of oxygen and forms hydrogen peroxide, in addition to the lactone. A second enzyme, called peroxidase, catalyzes the reaction of hydrogen peroxide with colorless compounds to create a colored product, which is quantified with a simple spectrophotometer. What are the circumstances required to make this possible? Aside from allowing the detection of smaller quantities of glucose, what advantage does glucose oxidase offer over the Fehling's reagent for measuring blood glucose?

Azathioprine is an immunosuppressant drug used in kidney transplants. In vivo, azathioprine is metabolized to the hypoxanthine analog 6- mercaptopurine, which is then converted into 6-mercaptopurine ribose monophosphate, the active form of the drug. 6- Mercaptopurine ribose monophosphate also inhibits de novo purine synthesis, reducing uric acid levels in the blood and urine. However, when administered to Lesch – Nyhan patients, there was no effect on uric acid levels. Explain why this is the case.

Suppose you discover that bacteria that express OmpF have concentrations of <0.1uM of ciprofloxacin in the cytosol but those without this substance have concentrations of up to 50uM in the cytosol by the time they die.  What does this tell you about the function of the isolated substance, OmpF, which you suspected from the start was related to quinolone resistance?  How is the OmpF structure optimized for this function? Which amino acids in OmpF are suited to each aspect of its function? The structure of ciprofloxacin is shown below for reference.

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…

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.

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.

Phosphoglycerate-mutase catalyses the isomerization of 3-phospho-glycerate to 2-phospho-glycerate. The enzyme has a molecular weight of 28,556 g/mol. In assays using 1 milligram of enzyme per assay the Km for 3-phosphoglycerate was 1.2 x 10^-3M and the Vmax was 840 μmole min^-1. What would be the Vmax in units of mole s^-1?

You generate mutants in the metabolic pathway for starlase. You conduct some complementation tests (after testing for dominance of course) and come up with the following results: 1 2 4 6 1 + + + 2 + 3 + 4 + 5 6 a. How many complementation groups are there? [Select] b. You conduct some additional experiments to elucidate the starlase metabolic pathway. Your results are shown below. Use this information alongside information from the complementation table above to place the intermediates in the correct order on the pathway. (HINT: use the complementation groups from the table above to help you consolidate information on the tables below. Reference practice question 3 from today's lecture for help). Addition to minimal medium Mutant None starlase P 1 + + 2 + + 4 + + 5 + 6 Precursor --> [ Select ] [ Select ] [ Select ] --> starlase c. Mutant 4 has a loss-of-function mutation for which enzyme in the starlase synthesis pathway? [ Select ] E1 E2 ЕЗ Е4 Precursor > Intermediate 1→ Intermediate…

The ABO blood group antigens are the terminal sugars covalently linked to the end of the glycolipid in the red blood cellmembrane. The H antigen is the precursor of the A and Bantigens. Individuals with type A blood produce a gene thatcodes for an enzyme that adds N-acetylgalactosamine in ana(1,3) linkage to the Gal* residue in the H antigen. Type Bblood requires that an enzyme add a d-a galactose in an a(1,3) linkage to the Gal*. Draw the structures of the A andB antigens.

Indicate the presence of the the B galactosidase and permease. Please explain and follow the instructions in the picture