### Dihydrofolate Reductase (DHFR) and Methotrexate (MTX)**Introduction**Dihydrofolate reductase (DHFR) is an enzyme that reduces 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate, a step crucial in the biosynthesis of thymidine. Methotrexate (MTX), a structural analogue of dihydrofolate, inhibits this enzyme. MTX has been employed in cancer therapies due to its inhibition of DHFR, which restricts thymidine production needed for cell division. This inhibition impedes the multiplication of cancerous cells.**Objective**Using the provided data of DHFR activity in the presence of MTX, the goals are to:1. Determine the inhibition mechanism of MTX and the maximum reaction rate.2. Calculate the inhibition constant \(K_i\) if the \(K_m\) for DHFR is 0.100 µM.**Data Table**The table below displays the reaction rate (in mMs\(^-1\)) of DHFR with varying concentrations of DHF and MTX:| [DHF](mM) | Rate w/ 50 nM MTX (mMs\(^-1\)) | Rate w/ 100 nM MTX (mMs\(^-1\)) | Rate w/ 200 nM MTX (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 | 7.38 |**Analysis**- **Step 1:** Determine the inhibition mechanism - Compare the rates of reaction at different concentrations of MTX. - Analyze how the inhibition changes based on varying concentrations of DHF. - Identify patterns that suggest whether MTX acts as a competitive, non-competitive, or uncompetitive inhibitor. - **Step 2:** Calculate the inhibition constant \(K_i\) - Use the given \(K

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Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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*The enzyme glucose oxidase isolated from the mold Penicillium notatum catalyzes the oxidation of 3-D-glucose to D-glucono-6- lactose. This enzyme is highly specific for the ß anomer of In spite of this glucose and does not affect the a anomer. 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 3- and a-D- glucose. What are the circumstances required to make this possible? Aside from allowing the detection of smaller quan- tities of glucose, what advantage does glucose oxidase offer over non-enzymatic oxidizing agents like Tollens reagent? *Is B-D-glucosamine a reducing sugar?
A common procedure for determining the effectiveness of compounds as precursors of glucose in mammals is to starve the animal until the liverglycogen stores are depleted and then administer the compound in question. A substrate that leads to a net increase in liver glycogen is termed glucogenic, because it must first be converted to glucose 6-phosphate. Show by means of known enzymatic reactions which of the following substances are glucogenic.
Draw a schematic illustration of the hydrolysis of N-acetylphenylalaninamide catalyzed byalpha-chymotrypsin in which you indicate the important catalytic residues in the active site and how thesubstrate undergoes transformation to products through two different tetrahedral intermediates.
[1-14C]Ribose-5-phosphate is incubated with a mixture of purified transketolase, transaldolase, phosphopentose isomerase, phosphopentose epimerase, and glyceraldehyde-3-phosphate. Predict the distribution of radioactivity in the erythrose-4-phosphate and fructose-6-phosphate that are formed in this mixture.
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…
Based on the image below, select the correct statement HN Aspartate Rib-P Inosinate (IMP) IMP H₂O OOC-CH₂- COO NH Fumarate N 274 adenylosuccinate lyase Rib-P Adenylosuccinate GTP GDP + P₁ adenylosuccinate IMP synthetase NAD+ NADH + H+ dehydrogenase Figure 22-36 Lehninger Principles of Biochemistry, Sixth Edition ©2013 W. H. Freeman and Company H HN H -N Gin Rib P Xanthylate (XMP) NH₂ Rib P Adenylate (AMP) Glu ATP AMP + PP₁ XMP-glutamine amidotransferase HN H₂N O GTP is phosphorylated in the conversion of IMP to GMP GTP is phosphorylated in the convertion of IMP to AMP GTP is dephosphorylated in the conversion of IMP to GMP O GTP is dephosphorylated in the conversion of IMP to AMP Rib-P Guanylate (GMP) GMP
The mechanism of chymotrypsin can be viewed as a two-step process, acylation of the enzyme active site followed by a deacylation reaction. What accounts for the observed "burst" in rapid kinetic studies of the hydrolysis of N-acetyl-L-phenylalanine p-nitrophenyl ester by chymotrypsin? The rate of hydrolysis of the acyl-enzyme intermediate is faster than the rate of forming the acyl-enzyme intermediate. The rates of the acylation and deacylation reactions are equal. The rate of the acylation reaction is slower than the rate of the deacylation reaction. The rate of the acylation reaction is faster than the rate of the deacylation reaction.
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.
During glycolysis, the enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) catalyzes the oxidation and phosphorylation of glyceraldehyde 3-phosphate, forming 1,3-bisphosphoglycerate. This reaction, shown below, has a ΔG that is close to zero under cellular conditions. During gluconeogenesis, GAPDH catalyzes the reverse reaction, in which 1,3-bisphosphoglycerate is reduced and dephosphorylated. Given this information, which of the following conditions are most likely to directly alter the relative rates of the forward and reverse reactions? (select two answers) changes in the relative concentrations of glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate changes in the relative concentrations of NAD+ and NADH decreased cellular concentration of pyruvate increased cellular concentration of glucose regulation of GAPDH activity
Cholera is caused by a toxin produced by the bacterium Vibrio cholerae. Ultimately, the toxin leads to the activation of ion channels by Protein Kinase A (PKA), indirectly causing water to leave the cell (following ions out of the cell). This loss of electrolytes and water from intestinal cells into the intestinal lumen, produces the watery diarrhea characteristic of a V.cholera infection. Would you expect lower or higher levels of PKA in cells exposed to the cholera toxin? If you were to measure CAMP levels in these cells, what would you expect to find? Why? How might the cholera toxin modify G-proteins to result in these cAMP levels?
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.
Peroxidase is an enzyme found in many organisms, from plant to humans. The function of peroxidase is to break down hydrogen peroxide, which is a toxin produced as a byproduct when oxygen is produced during respiration. Myeloperoxidase is a protein found in neutrophils and catalyzes lipid peroxidation involved in immune defense. The overproduction of myeloperoxidase has been found to function as a mediator for tissue damage in inflammatory diseases. (8 points) a. Identify two examples of environmental factors that may impact myeloperoxidase activity. Describe how each of the environmental factors would affect the reaction rate of the enzyme. b. Predict the how researchers can use myeloperoxidase activity as oxidative stress biomarkers in patients with rheumatoid arthritis. Provide reasoning to justify your prediction.

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