Case 32
Inhibition of protein tyrosine phosphatase enzymes as a possible treatment for diabetes
Last modified 10 March 2009
Focus concept
Protein tyrosine phosphatases (PTPs) are investigated because of their ability to reverse the effects of protein tyrosine kinases (PTKs). Inhibitors of the PTP investigated in this study have insulin-mimetic effects and have potential in the treatment of diabetes.
Prerequisites
Enzyme kinetics and mechanisms of inhibition
Lineweaver-Burk analysis
Enzyme catalytic mechanisms
Background
Figure 32.1. There is a delicate balance between activating a receptor tyrosine kinase (which leads to the phosphorylation of specific tyrosines) and activating a protein tyrosine phosphatase, which removes the phosphates from the tyrosines (from Nature Reviews: Drug Discovery (2002) 1, p. 774.
Receptor tyrosine kinases are a family of transmembrane proteins that are activated by extracellular signaling molecules. In the absence of ligand binding, most receptor tyrosine kinases exist as monomers. The signaling molecules act as ligands to dimerize the extracellular domain of the receptor. This causes a conformational change that activates the C-terminal protein kinase domain located on the cytoplasmic side of the membrane. The tyrosine kinase catalyzes the autophosphorylationof specific tyrosine residues located on the intracellular domain of the receptor. These phosphorylated tyrosines serve as docking sites for cytosolic proteins which bind to the phosphorylated tyrosines and then put into motion a cascade of intracellular reactions that leads to changes in cellular growth and differentiation. Many growth factors act as ligands and exert their effects using this mechanism. Insulin is another example of a ligand that binds to a receptor tyrosine kinase. Its receptor differs slightly from the growth factor receptors in that it is a dimer in the unliganded state. Binding of insulin is still required, however, to cause the conformational change necessary to activate the tyrosine kinase domain.
Protein-tyrosine phosphatase (PTP) enzymes have the ability to reverse the effects of the activators of the protein tyrosine kinases because the phosphatase enzyme catalyzes the hydrolysis of phosphates from the phosphotyrosines on the receptor. Thus they too are important in cellular regulation. There is a delicate balance in the cell between activation of the receptor tyrosine kinases by phosphorylation and deactivation of the receptor catalyzed by the protein tyrosine phosphatases (Figure 32.1). Inhibitors of the protein tyrosine phosphatases have the effect of potentiating the action of the kinases. In the absence of phosphatase activity, the receptor tyrosine kinase has a longer period of sustained activity.
Figure 32.2. Opposite actions of protein tyrosine kinases (top reaction) and protein tyrosine phosphatases (bottom reaction).
For example, an inhibitor of the protein tyrosine phosphatase PTP1B acts to potentiate the activity of the insulin receptor (a protein tyrosine kinase) and in so doing, acts as an insulin mimetic. In clinical trials, inhibitors of PTP1B have been shown to be effective at treating diabetes.
As mentioned above, protein tyrosine phosphatases catalyze the hydrolysis of phosphate groups from specific phosphotyrosyl residues. PTP enzymes have an essential cysteine in the catalytic site of the enzyme that is essential for activity. During the process of the removal of the phosphate from its substrate, a transient covalent bond is formed between the thiol group of the cysteine and the phosphate group. Oxidation of the essential cysteine removes the sulfhydryl group and renders the enzyme inactive; therefore during the purification process, dithiothreitol (DTT), a reducing agent, was added to buffers to keep the essential cysteine in its reduced form.
In this study, the investigators studied the ability of vanadate and pervanadate to inhibit a protein tyrosine phosphatase called PTP1B. The PTP1B enzyme studied here was a cytosolic enzyme consisting of 321 amino acids with a calculated molecular weight of 37366 Da. In order to assay the enzymatic activity, the investigators used an artificial substrate, fluorescein diphosphate (FDP). Removal of a phosphate group from FDP by the PTPIB enzyme produces the product fluorescein monophosphate (FMP), which absorbs light at 450 nm; thus the rate of reaction can be measured by monitoring the rate of formation of product by spectroscopic methods. The reaction is shown is Figure 32.3. The structure of vanadate, and its comparison to phosphate, is shown in Figure 32.4.
Figure 32.3. PTP1B-catalyzed hydrolysis of phosphate from fluorescein diphosphate (FDP) to produce fluorescein monophosphate (FMP); the formation of the product can be monitored spectroscopically at 450 nm.
Figure 32.4. Structural similarities exist between phosphate and vanadate.
The investigators measured the activity of the PTP1B enzyme in the presence and in the absence of 4.0 M vanadate. The reaction was carried out as shown in Figure 32.3. The concentration of PTP1B was 400 ng/mL. The data are shown in Table 32.1.
Table 32.1: Hydrolysis of FDP by PTP1B in the presence and the absence of vanadate.
[FDP], M / velocity, nM/s (w/o vanadate) / velocity, nM/s (w/ vanadate)6.67 / 5.7 / 0.71
10.0 / 8.3 / 1.06
20.0 / 12.5 / 2.04
40.0 / 16.7 / 3.70
100.0 / 22.2 / 8.00
200.0 / 25.4 / 12.5
The investigators also studied the ability of pervanadate to inhibit PTP1B activity. “Pervanadate” is the name given to a family of compounds that are formed when vanadate reacts with hydrogen peroxide. The structure of one of the complexes formed is shown in Figure 32.5.
Figure 32.5. Structure of the monoperoxo complex of vanadate.
Questions
1.The investigators compared the ability of vanadate and pervanadate to inhibit the activity of PTP1B. They added enzyme and substrate, then added inhibitor. They found that the inhibition by vanadate could be reversed if EDTA (a chelating agent that binds both vanadate and pervanadate) was added, but the inhibition by pervanadate was only partially reversed by EDTA. In general, what does this tell you about the mode of action of the two inhibitors?
2.Construct a Lineweaver-Burk plot using the data provided in Table 32.1. Calculate KM and Vmax for PTP1B in the absence and in the presence of vanadate. Show your calculations.
3.What kind of inhibitor is vanadate? Explain, using the data in the Lineweaver-Burk plot. Calculate α or α’ (or both), whatever is appropriate, then determine KI or KI’ (or both, again, whatever is appropriate). Calculate kcat in the absence of inhibitor. Show your calculations.
4.Using the information in the Background and in the Lineweaver-Burk plot, explain how vanadate inhibits the activity of PTP1B.
5.An alternative way to calculate KI for an inhibitor is to measure the velocity of the enzyme-catalyzed reaction in the presence of increasing amounts of inhibitor and a constant amount of substrate. These data are shown in Table 32.2 for a substrate concentration of 6.67 μM. For each concentration of inhibitor, calculate , then plot vs. [I]. Determine KI from the slope of the plot.
Table 32.2: Velocity of the PTP1B reaction in the presence of varying amounts of vanadate.
[vanadate], M / velocity, nM/s0.0 / 5.70
0.20 / 3.83
0.40 / 3.07
0.70 / 2.35
1.0 / 2.04
2.0 / 1.18
4.0 / 0.71
Elucidation of the mechanism by which pervanadate inhibits PTP1B
6.Refer to your answer to Question 1. The investigators were interested in determining the mechanism by which pervanadate inhibits PTP1B. Because the active site contains an essential Cys, the authors of this study considered how the Cys residue might be altered in such a way so that catalysis could not occur. They reasoned that Cys could be oxidized to form four possible products: Either a disulfide bond could form (-S-S-), the Cys sulfhydryl could form sulfenic acid (-SOH), or sulfinic acid (-SO2H) or sulfonic (cysteic) acid (-SO3H). So after carrying out the inhibition studies described in Question 1, the investigators isolated the PTP1B enzyme and subjected the enzyme to analysis by mass spectrometry. They found that the mass of PTPIB increased by 48 g/mol in the presence of pervanadate. Propose a hypothesis in which you explain the mechanism of inhibition of PTP1B by pervanadate that is consistent with the data.
Physiological importance of the PTP1B enzyme
7.Another group of investigators worked with a strain of mice in which the PTP1B gene was rendered nonfunctional. They worked with three groups of mice. The PTP1B+/+ mice inherited a normal gene from each parent; the PTP1B+/- mice inherited a functional gene from one parent and a nonfunctional gene from the other parent, and the PTP1B-/- mice inherited a non-functional gene from each parent. The blood glucose concentration (panel A) and the blood insulin concentration (panel B) of the two groups of mice were measured under both fed (dark bars) and fasting (light bars) conditions. A * indicates a p value of 0.06 and a ** indicates a p value less than 0.01. What is your interpretation of these data?
Figure 32.6. Determination of blood glucose (A) and insulin (B) concentrations in wild type and PTP1B-deficient mice. The dark bars represent fed conditions and the light bars are fasting conditions (taken from Elchebly, et al., 1999).
References
Huyer, G., Liu, S., Kelly, J., Moffat, J., Payette, P., Kennedy, B., Tsaprailis, G., Gressner, M. J., and Ramachandran, C. (1997) “Mechanism of Inhibition of Protein-tyrosine Phosphatases by Vanadate and Pervanadate” J. Biol. Chem. 272, pp. 843-851.
Elchebly, M., Payette, P., Michaliszyn, E., Cromlish, W., Collins, S., Loy, A. L., Normandin, D., Cheng, A., Himmis-Hagen, J., Chan, C.-C., Ramachandran, C., Gresser, M. J., Tremblay, M. L., and Kennedy, B. P. (1999) “Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene” Science283, 1544-1548.
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