o 1. Protein tyrosine phosphatase enzymes are important regula- tory enzymes. Clinical trials show that inhibitors of these en- zymes can be important means for treating certain diseases, e.g., diabetes. Phosphatase enzymes catalyze removal of the phos- phoryl group from tyrosine residues of specific proteins convert- ing the phosphoryl group, i.e., PO3²-, into a phosphate through hydrolysis. As an example of a phosphatase enzyme, below are data collected to characterize protein tyrosine phosphatase-1B (PTP1B), an important enzyme regulating part of the pathway for uptake of glucose into cells. The activity was measured in the presence and absence of vanadate (VO43-), an inhibitor of the enzyme. An artificial substrate, fluores- cein diphosphate (FDP), shown on the right, was used in the experiments. Upon hydrolysis FDP forms the product fluorescein monophosphate (FMP) that absorbs light at 450 nm, allowing activity to be measured spectrophotometrically. The results are provided in the table below: Vo without vanadate (VO4³-) Vo with vanadate (VO4³-) (nm. s¹) 5.7 8.3 12.5 16.7 22.2 25.4 [FDP] (µM) 6.67 10 20 40 100 200 (nM. s-¹) 0.71 1.06 2.04 3.70 8.00 12.5 (a) Construct a Lineweaver-Burk plot using the data provided. Calculate KM and Vmax for PTP1B in the absence and in the presence of vanadate. (b) What kind of an inhibitor is vanadate (VO4³-)? Explain the criteria that define this type of inhibition and explain why structurally one would anticipate this type of inhibition.
o 1. Protein tyrosine phosphatase enzymes are important regula- tory enzymes. Clinical trials show that inhibitors of these en- zymes can be important means for treating certain diseases, e.g., diabetes. Phosphatase enzymes catalyze removal of the phos- phoryl group from tyrosine residues of specific proteins convert- ing the phosphoryl group, i.e., PO3²-, into a phosphate through hydrolysis. As an example of a phosphatase enzyme, below are data collected to characterize protein tyrosine phosphatase-1B (PTP1B), an important enzyme regulating part of the pathway for uptake of glucose into cells. The activity was measured in the presence and absence of vanadate (VO43-), an inhibitor of the enzyme. An artificial substrate, fluores- cein diphosphate (FDP), shown on the right, was used in the experiments. Upon hydrolysis FDP forms the product fluorescein monophosphate (FMP) that absorbs light at 450 nm, allowing activity to be measured spectrophotometrically. The results are provided in the table below: Vo without vanadate (VO4³-) Vo with vanadate (VO4³-) (nm. s¹) 5.7 8.3 12.5 16.7 22.2 25.4 [FDP] (µM) 6.67 10 20 40 100 200 (nM. s-¹) 0.71 1.06 2.04 3.70 8.00 12.5 (a) Construct a Lineweaver-Burk plot using the data provided. Calculate KM and Vmax for PTP1B in the absence and in the presence of vanadate. (b) What kind of an inhibitor is vanadate (VO4³-)? Explain the criteria that define this type of inhibition and explain why structurally one would anticipate this type of inhibition.
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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![**Protein Tyrosine Phosphatase Enzymes**
1. **Introduction:**
Protein tyrosine phosphatase enzymes are crucial regulatory enzymes. Clinical trials indicate that inhibitors of these enzymes can be potential treatments for diseases such as diabetes. These enzymes catalyze the removal of the phosphoryl group from tyrosine residues, converting the phosphoryl group (PO₃²⁻) into a phosphate through hydrolysis. This process is vital for various cellular functions.
2. **Experiment Overview:**
The experiment focuses on protein tyrosine phosphatase-1B (PTP1B), an enzyme that regulates pathways related to glucose uptake into cells. Vanadate (VO₄³⁻) was used as an enzyme inhibitor, and activity was measured using the substrate fluorescein diphosphate (FDP). Hydrolysis of FDP produces fluorescein monophosphate (FMP), which absorbs light at 450 nm, allowing for spectrophotometric measurement of enzyme activity.
3. **Results:**
The table below displays enzyme activity (V₀) with varying concentrations of FDP, both in the absence and presence of vanadate:
| [FDP] (μM) | V₀ without vanadate (VO₄³⁻) (nM·s⁻¹) | V₀ with vanadate (VO₄³⁻) (nM·s⁻¹) |
|------------|--------------------------------------|-----------------------------------|
| 6.67 | 5.7 | 0.71 |
| 10 | 8.3 | 1.06 |
| 20 | 12.5 | 2.04 |
| 40 | 16.7 | 3.70 |
| 100 | 22.2 | 8.00 |
| 200 | 25.4 | 12.5 |
4. **Tasks:**
(a) Construct a Lineweaver-Burk plot using the data provided. Calculate \( K_M \) and \( V_{max} \) for PTP1B without and with vanadate.
(b) Explain what kind of inhibitor vanadate (VO₄³⁻) is. Provide criteria that define this](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F7e3218aa-1b65-42a2-9071-7edb43c3a555%2F63c9f8ef-0500-4c89-bf0e-2f5d4fea92e0%2F4qlnen9_processed.png&w=3840&q=75)
Transcribed Image Text:**Protein Tyrosine Phosphatase Enzymes**
1. **Introduction:**
Protein tyrosine phosphatase enzymes are crucial regulatory enzymes. Clinical trials indicate that inhibitors of these enzymes can be potential treatments for diseases such as diabetes. These enzymes catalyze the removal of the phosphoryl group from tyrosine residues, converting the phosphoryl group (PO₃²⁻) into a phosphate through hydrolysis. This process is vital for various cellular functions.
2. **Experiment Overview:**
The experiment focuses on protein tyrosine phosphatase-1B (PTP1B), an enzyme that regulates pathways related to glucose uptake into cells. Vanadate (VO₄³⁻) was used as an enzyme inhibitor, and activity was measured using the substrate fluorescein diphosphate (FDP). Hydrolysis of FDP produces fluorescein monophosphate (FMP), which absorbs light at 450 nm, allowing for spectrophotometric measurement of enzyme activity.
3. **Results:**
The table below displays enzyme activity (V₀) with varying concentrations of FDP, both in the absence and presence of vanadate:
| [FDP] (μM) | V₀ without vanadate (VO₄³⁻) (nM·s⁻¹) | V₀ with vanadate (VO₄³⁻) (nM·s⁻¹) |
|------------|--------------------------------------|-----------------------------------|
| 6.67 | 5.7 | 0.71 |
| 10 | 8.3 | 1.06 |
| 20 | 12.5 | 2.04 |
| 40 | 16.7 | 3.70 |
| 100 | 22.2 | 8.00 |
| 200 | 25.4 | 12.5 |
4. **Tasks:**
(a) Construct a Lineweaver-Burk plot using the data provided. Calculate \( K_M \) and \( V_{max} \) for PTP1B without and with vanadate.
(b) Explain what kind of inhibitor vanadate (VO₄³⁻) is. Provide criteria that define this
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Step 1: Relation between Michaelis Menten equation and Lineweaver Burk Plot
VIEWStep 2: Lineweaver Burk plot for the given enzyme in presence of inhibitor
VIEWStep 3: Calculation of Km and vmax from the straight line equation in LB plot
VIEWStep 4: Type of inhibition exerted by vanadate
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