Vanadium: The Versatile Metal - American Chemical Society

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Chapter 7

Enhancement of Insulin Action by bis(Acetylacetonato)oxovanadium(IV) Occurs through Uncompetitive Inhibition of Protein Tyrosine Phosphatase-1B 1,*

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Marvin W. Makinen , Stephanie E. Rivera , Katherine I. Zhou , and Matthew J. Brady 2

1Department of Biochemistry and Molecular Biology, Gordon Center for Integrative Science, The University of Chicago, 929 East 57 Street, Chicago, IL 60637 Department of Medicine and Committee on Molecular Metabolism and Nutrition, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637 th

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We have examined the influence of bis(acetylacetonato)oxovanadium(IV) [VO(acac) ] on the catalytic activity of protein tyrosine phosphatase-1B (PTP1B). In the presence of p-nitrophenylphosphate as the substrate, VO(acac) exhibited mixed inhibition. However, VO(acac) exhibited uncompetitive inhi bition of the enzyme with the undecapeptide substrate D A D EpYLIPQQG, in which the sequence corresponds to residues 988-998 of the epidermal growth factor receptor and pY indicates the phosphotyrosine residue. These results are consistent with our earlier observations, on the basis of phos photyrosine immunoblots, showing that VO(acac)2 potentiates tyrosine phosphorylation of the insulin receptor synergistically with insulin. Because uncompetitive inhibitors of PTP1B have not been described heretofore, we discuss the importance of uncompetitive inhibition with respect to design of inhibitors of the enzyme for therapeutic purposes. 2

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© 2007 American Chemical Society

In Vanadium: The Versatile Metal; Kustin, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Introduction Because impaired insulin action is the underlying cause of type 2 diabetes, the search for suitable targets of pharmacologic agents to alleviate the patho physiology of this condition has necessarily focused on the insulin signaling pathway (7-5). Activation of the insulin receptor (IR) on the cell surface by binding of insulin initiates phosphorylation of tyrosine residues through the autophosphorylative and tyrosine kinase activities of the receptor, culminating in the translocation of glucose into the cell from the blood stream by glucose transporter systems (4-6). While a series of further downstream phosphorylative events continues the signaling events to result in glycogen formation or fatty acid synthesis, the activated IR must be returned to a basal state to respond anew to the binding of insulin. Protein tyrosine phosphatase-IB (PTP1B) catalyzes the hydrolysis of phosphotyrosine containing peptides (7-9), and, as a member of the protein tyrosine phosphatase superfamily of enzymes (10), is thought to have a major role in deactivation of the IR (//, 12). Because there are over 100 known members of this enzyme superfamily, sharing common structural motifs and similar active site residues involved in signal transduction and cellular processes such as growth, differentiation, and proliferation, inhibition of PTP1B must be not only potent but also highly specific. In earlier studies of the insulin enhancing effects of organic chelates of the vanadyl ( V 0 ) ion to facilitate glucose uptake by cultured 3T3-L1 adipocytes, we observed that the behavior of Ws(acetylacetonato)oxovanadium(IV) [VOacac) ] was synergistic with added insulin, in contrast to that of Ws(maltolato)oxovanadium(IV) [VO(malto) ] (13,14). This observation was made on the basis of phosphotyrosine immunoblots of cell lysates showing a dose-dependent increase in phosphotyrosine levels of the IR and the insulin receptor substrate-1 (IRS-1) after the cells were challenged for glucose uptake in the presence of V0 -chelates. Because VO(acac) elicits an increase in the phosphotyrosine content of the IR and IRS-1 in the presence of wortmannin, a specific inhibitor of phosphatidyl inositol 3-kinase, we concluded that VO(acac) acts directly on one or more enzymes that regulate the phosphotyrosine content of the IR (14). To this end, we have investigated the influence of VO(acac) on the catalytic properties of human recombinant PTP1B. Our results demonstrate that V O (acac) acts as a mixed inhibitor of PTP1B when p-nitrophenylphosphate (pNPP) is employed as the substrate. However, in the presence of the phosphotyrosine containing undecapeptide analog of the epidermal growth factor receptor, resi dues 988 - 998 having the amino acid sequence D A D E p Y L I P Q Q G , VO(acac) acts as an uncompetitive inhibitor. Not only is the observation of uncompetitive inhibition unusual, but also the results explain the synergism of VO(acac) with insulin and the lack of insulin synergism with VO(malto) . Because no uncompetitive inhibitor of PTP1B has been reported in the literature, to our 2+

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84 knowledge, we discuss the importance of these observations with respect to inhibitor design for therapeutic purposes.

Experimental Procedures General Crystalline VO(acac) was purchased from Sigma-Aldrich (Milwaukee, WI 53209) and used without further purification. The di-sodium salt of pNPP and D,L-dithiothreitol were obtained from Fluka Chemical Company (Milwaukee, WI 53233) and used directly. A l l other chemicals were of analytical reagent grade, and doubly distilled, deionized water was used throughout. The D N A corresponding to the soluble portion of wild type human PTP1B (residues 1-321), kindly provided by Professor Z.-Y. Zhang, was cloned into E. coli BL21(DE3) cells following the protocol described from the Zhang labora tory (8,15). Engineered cells were grown in liquid Luria-Bertani medium at 37 °C for 12 hours following induction with isopropyl-l-thio-p-galactoside; lysis of cells was achieved by several freeze-thaw cycles using liquid nitrogen followed by 12 - 15 cycles of sonication, each cycle lasting 20 - 30 s. For sonication the cell suspension was kept submerged in ice-water to prevent heating. The cell suspension was then centrifuged at 5000 x g for 20 min, the supernatant carefully decanted and mixed with pre-swollen CM-Sephadex C50 according to Zhang and co-workers (15). Elution of the enzyme from CM-Sephadex C50 was achieved with a 0.04 M - 0.5 M gradient of NaCl. The pooled fractions containing enzyme determined on the basis of the optical density at 280 nm and the presence of catalytic activity were pooled and concentrated with an Ultrafree centrifugal filter device with a 10,000 molecular weight cut-off (Millipore Corp., Bedford, M A 01730).


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Kinetic Studies Initial velocity data were collected with a Cary 15 spectrophotometer modi fied by On-Line Instrument Systems, Inc. (Bogart, G A 30622) for micropro cessor-controlled data acquisition and equipped with an efficient mixing device for kinetic studies (16). Hydrolysis of the substrate pNPP was monitored at 349 nm, experimentally determined to yield the largest change in absorption for monitoring the reaction at pH 5, the pH optimum for enzyme activity (8). The (^substrate - E ) difference extinction coefficient was 1911 M~ cm" . Only freshly prepared stock solutions of pNPP, dissolved in 0.1 M NaCl buffered to ,

product


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85 pH 5.0 with 0.01 M sodium acetate, were used. The pNPP concentration was varied from (0.25 - 5) x A ^ . Crystalline VO(acac)2 was directly dissolved in 0.1 M sodium chloride buffered to pH 5 with 0.01 M acetate and extensively purged with N prior to use. The highest VO(acac) concentration in reaction mixtures was 90.0 x 10" M . Kinetic data were analyzed with use of Origin 5.0, released by Microcal Software, Inc. (Northampton, M A 01060). 2

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The influence of VO(acac) on the enzyme-catalyzed reaction was also evaluated with use of the Protein Tyrosine Phosphatase 1B Assay Kit (Calbiochem, La Jolla, C A 92039) based on colorimetric Malachite Green detection of inorganic phosphate released as the product. In this reaction, however, the sub strate is the phosphotyrosine containing undecapeptide D A D E p Y L I P Q Q G where pY represents the phosphotyrosine residue. The amino acid sequence of this peptide corresponds to residues 988-998 of the epidermal growth factor receptor. Reaction mixtures were set up at 30 °C in 96-welI plates and the amount of inor ganic phosphate released as product was measured at 620 nm with a Tecan Safire microplate scanner (Tecan A G , CH-8708 Mannedorf, Switzerland) running on XFluor software. Assay measurements were made in triplicate, and reaction conditions at ambient temperature were established to ensure phosphate release within the linear portion of the standard curve for measurement of phosphate concentration. A n incubation time of 30 minutes was employed for the reaction prior to quenching of the reaction and addition of Malachite green for color formation.


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Results Steady-State Kinetic Assays For the steady-state kinetic parameters k and K , initial velocity data for the hydrolysis of pNPP catalyzed by PTP1B yielded values of 36.5 + 2.0 s" and 4.2 ± 0 . 1 x 10~ M , respectively, at ambient room temperature (-22 ° C ) . While the value of K at pH 5 agrees well with that of ~ 4 x 10~ M reported by Zhang and coworkers (#), our value of k is higher. Zhang et al. report a value o f - 3 7 s" at 37 °C, requiring that the value at 22 °C would be lower. Our experiments were carried out with use of an efficient mixing device (16) with mixing time of < 6 s at room temperature. Micro cuvettes were employed in experiments reported by Zhang and co-workers with no mention of the stirring assembly for initiating the reaction (8). Inefficient mixing causes an apparent decrease in initial velocity, and this decrease may be in part the origin of the difference in * values. cat

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86 Figure 1 illustrates the inhibitory behavior of VO(acac) in hydrolysis of pNPP catalyzed by PTP1B. As in experiments carried out in the absence of V O (acac) , the Lineweaver-Burk plot shows evidence for substrate inhibition. To best evaluate the influence of VO(acac) , therefore, the data at high substrate concentrations, represented with open symbols, were not used to estimate slope and intercept values. The results indicate that VO(acac) acts as a mixed inhibi tor. From these results, the K\ of this organic chelate of V 0 is estimated to lie in the 15 - 20 x 10~ M range. In contrast, VO(malto) is reported to act as a competitive inhibitor under steady-state conditions with use of the fluorescent substrate 6,8-difluoro-4-methylumbeliferyl phosphate (17). O f special interest in the study by Peters and coworkers is the observation that the hydrated V 0 cation stripped of its organic ligands acts as the competitive inhibitor (17). 2

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1/[pNPP] (mM* ) Figure 1. Double reciprocal

plots of initial velocity data collected for the

hydrolysis of pNPP catalyzed by PTP1B at pH 5. VO(acac)2 concentrations indicated adjacent to the line of the corresponding

least-squares

are

linear fit to

each data set.

Malachite Green Colorimetric Assays Figure 2 illustrates a double reciprocal plot of phosphate release from the phosphotyrosine containing undecapeptide D A D E p Y L I P Q Q G , as measured with


87 the Malachite Green colorimetric method. The enzyme commercially supplied with the assay kit similarly showed a tendency towards substrate inhibition. As with use of pNPP as the substrate, data at high substrate concentrations were not used to calculate the slope and intercept of the straight-line graphs. In sharp contrast to the use of pNPP as the substrate, VO(acac)2 exhibits uncompetitive inhibition against the undecapeptide substrate. From these results, the K\ of the V0 -chelate was estimated to lie in the 5 - 8 x 10 M range. It is of consider able interest to note that no uncompetitive inhibitor of PTP1B has been previ ously described. Downloaded by UNIV OF MELBOURNE on October 14, 2014 | http://pubs.acs.org Publication Date: August 30, 2007 | doi: 10.1021/bk-2007-0974.ch007

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[VO(acac) ] 2

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Figure 2. Comparison

0.12

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of the influence of VO(acac)2 on FTP IB

hydrolysis of the phosphotyrosine

containing

evaluated with use of a Malachite

Green colorimetric

concentrations

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0.04 0.08 1/ [DADEpYLIPQQG] (M" X 10 )

catalyzed

undecapeptide substrate, as method.

VO(acac)2

are indicated on the right for each double-reciprocal

plot.

Discussion In contrast to our results in Figure 1, illustrating mixed inhibition of PTP1B by VO(acac) , Peters and co-workers observed competitive inhibition of PTP1B 2


88 by VO(maIto) (17). While a small molecule, organic phosphate ester was used as the substrate in the study by Peters and co-workers, as for the results in Figure 1, the significant difference lies in the observation that the hydrated V 0 ion is the competitive inhibitor when VO(malto)2 is used. This result, as noted by Peters et al. (17), indicates that the V 0 ion is stripped of its organic ligands upon binding to the enzyme. Since the pattern of inhibition in Figure 1 requires a mixture of classical competitive and noncompetitive inhibition, we cannot exclude the possibility that a small fraction of VO(acac) is dissociated, releasing some of the V 0 ion to act as a competitive inhibitor in the active site, as in the case of VO(malto) . However, because the patterns in Figures 1 and 2 are so dif ferent from each other and because VO(acac) is significantly more stable in aqueous solution than VO(malto) (14,18), we conclude that the VO(acac) com plex added to the enzyme reaction mixture remains intact and accounts for the noncompetitive and uncompetitive inhibitory behavior illustrated in Figures 1 and 2, respectively, dependent on the substrate. This observation requires that VO(acac) binds as an organic chelate, either as an intact complex or possibly as a hemi-liganded [VO(acac)] complex. 2

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Hydrolysis of a phosphotyrosine containing polypeptide substrate catalyzed by PTP1B requires that the phosphate moiety esterified to the hydroxyl group of the tyrosine side chain is positioned into the active site for nucleophilic attack by Cys-215 (8,15,19). The steric properties of the phosphate group of pNPP, esteri fied to the hydroxyl group of p-nitrophenol, are essentially identical, ensuring a similar stereochemical approach to the nucleophilic sulfhydryl group. The obser vation through Figures 1 and 2 that the pattern of inhibition is dependent on substrate structure requires, therefore, that the site occupied by VO(acac) as a noncompetitive inhibitor in Figure 1 differs from that occupied by VO(acac) as an uncompetitive inhibitor in Figure 2. This conclusion is supported by estimates of the inhibitor constant of VO(acac) as a noncompetitive inhibitor (15-20 x 10 M ) and as an uncompetitive inhibitor (5-8 x 10~ M ) extracted from the graphical plots. While VO(acac) as a noncompetitive inhibitor may bind near the pNPP substrate, the more extended steric volume of the undecapeptide substrate probably blocks this binding site, forcing the V0 -chelate to find a different binding location on the enzyme, resulting in uncompetitive inhibition. While definition of the binding site of VO(acac) on the enzyme as an uncompe titive inhibitor awaits X-ray crystallographic analysis, its identification through Figure 2 suggests that this site may be important as a focus of inhibitor design for therapeutic purposes. Because the structural relationships of backbone and side chain atoms located in the immediate vicinity of catalytically active residues of PTP1B are well defined through X-ray crystallography, the focus of inhibitor design studies has been entirely directed towards antimetabolites that compete with the sub strate in the active site (1,2,19-22). This approach constitutes a large fraction of 2

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89 the literature on structure based drug design in general. It has been demonstrated, however, that in open systems, in which there is constant input of substrate and removal of products, as in cells, this strategy is unlikely to be effective (23,24). A n approach based on uncompetitive inhibition is far superior when the efficacy of inhibition of metabolic reactions is considered. With respect to kinetic rela tionships, the differences between these two approaches and the greater effect iveness of an uncompetitive inhibitor over competitive inhibitors are illustrated in Figure 3. Figure 3 compares the inhibition patterns of competitive and uncompetitive inhibitors and their characteristic hyperbolic plots of relative reaction velocity vs. substrate concentration. The decrease in reaction velocity at any given sub strate concentration is also illustrated according to the ratio of the concentration of the inhibitor to the value of the inhibitor binding constant. The hallmark of competitive inhibition is that the inhibitor bound in the active site can be displaced by substrate, particularly when there is constant input of substrate into the system, as occurs in the cell, with only a fixed amount of inhibitor. Under these conditions, competitive inhibitors cannot be expected to provide effective, long-term inhibition. On the other hand, the kinetic scheme for uncompetitive inhibition shows that the inhibitor has affinity for only the enzyme-substrate complex. Because open systems are distinguished by constant input of substrate, correspondingly, with build-up of enzyme-substrate complex, the inhibition gains in effectiveness in the long-term because there is an increasing amount of the enzyme-substrate complex as the only target of the inhibitor. For competitive inhibition with a fixed inhibitor concentration, the relative velocity increases as the substrate concentration increases because the inhibitor is displaced. On the other hand, for a fixed amount of an uncompetitive inhibitor, the relative velocity quickly reaches a plateau value as the substrate concentration increases, resulting in sustained inhibition as a long-term effect. The different patterns illustrated by the hyperbolic plots in Figure 3 for com petitive and uncompetitive inhibition help to explain our observations that the insulin-enhancing capacity of VO(acac)2 is synergistic with insulin while that of VO(malto)2 is not. In measurements of glucose uptake by cultured 3T3-L1 adi pocytes, we observed that the level of the phosphorylated form of the IR, detected on the basis of phosphotyrosine-specific antibodies, was greater when elicited by VO(acac) and insulin together in the incubation medium than when elicited by either VO(acac)2 or insulin alone (14). According to Figure 3, a ter nary ESI complex is formed through the action of VO(acac) as an uncompeti tive inhibitor, in which the substrate is the (tyrosine) phosphorylated form of the IR. 2

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Designating phosphorylated IR as pY(IR) and the ESI ternary complex as [VO(acac)2: PTP1B : pY(IR)], we can readily see that addition of insulin gener ates pY(IR) which becomes "trapped" in the form of the ternary complex



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Figure 3. Comparison of reaction schemes and hyperbolic plots of relative reaction velocity vs. substrate concentration for competitive and uncompetitive inhibition of an enzyme catalyzed reaction.

[VO(acac) : PTP1B : pY(IR)] on top of that formed in the presence of V O (acac) alone. On the other hand, because VO(malto)2 is effectively a competi tive inhibitor (77), addition of insulin to the incubation medium containing this V0 -chelate generates more pY(IR) that simply displaces V 0 from the enzyme, allowing the increased quantity of pY(IR) to be hydrolyzed by PTP1B. Thus, as confirmed through experiment in the cell, the level of pY(IR), detected on the basis of phosphotyrosine-specific immunoblotting, is unchanged with increased addition o f insulin in the presence o f VO(malto)2 and remains essen tially identical to that detected in the presence of insulin alone (14). This very different pattern of uncompetitive inhibition arises because the inhibitor has affinity only for the enzyme-substrate complex. Because such com plexes are not usually described through X-ray structural analysis, design of an uncompetitive inhibitor of an enzyme is invariably more difficult than design of a competitive inhibitor. Nonetheless, the effort is likely to be more rewarding. As pointed out by Cornish-Bowden (23), an uncompetitive inhibitor of an enzyme that makes only a very small contribution to setting the flux of metabo lites through a pathway is much more likely to have a major pharmacological effect than a competitive inhibitor. 2

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Acknowledgments We thank Professor Z.-Y. Zhang for providing the D N A for overexpression of PTP1B. This work was supported by a grant of the National Institutes of Health (DK064772) awarded to M . J. B.

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Vanadium: The Versatile Metal - American Chemical Society

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