Kinetic characterization of the inhibition of protein tyrosine phosphatase-1B by Vanadyl (VO2+) chelates

Abstract

Protein tyrosine phosphatases (PTPases) are a prominent focus of drug design studies because of their roles in homeostasis and disorders of metabolism. These studies have met with little success because (1) virtually all inhibitors hitherto exhibit only competitive behavior and (2) a consensus sequence H/V-C-X₅-R-S/T characterizes the active sites of PTPases, leading to low specificity of active site directed inhibitors. With protein tyrosine phosphatase-1B (PTP1B) identifed as the target enzyme of the vanadyl (VO²⁺) chelate bis(acetylacetonato)oxidovanadium(IV) [VO(acac)₂] in 3T3-L1 adipocytes [Ou et al. J Biol Inorg Chem 10: 874-886, 2005], we compared the inhibition of PTP1B by VO(acac)₂ with other VO²⁺-chelates, namely, bis(2-ethyl-maltolato)oxidovanadium(IV) [VO(Et-malto)₂] and bis(3-hydroxy-2-methyl-4(1H)pyridinonato)oxidovanadium(IV) [VO(mpp)₂] under steady-state conditions, using the soluble portion of the recombinant human enzyme (residues 1-321). Our results differed from those of previous investigations because we compared inhibition in the presence of the nonspecific substrate p-nitrophenylphosphate and the phosphotyrosine-containing undecapeptide DADEpYLIPQQG mimicking residues 988-998 of the epidermal growth factor receptor, a relevant, natural substrate. While VO(Et-malto)₂ acts only as a noncompetitive inhibitor in the presence of either subtrate, VO(acac)₂ exhibits classical uncompetitive inhibition in the presence of DADEpYLIPQQG but only apparent competitive inhibition with p-nitrophenylphosphate as substrate. Because uncompetitive inhibitors are more potent pharmacologically than competitive inhibitors, structural characterization of the site of uncompetitive binding of VO(acac)₂ may provide a new direction for design of inhibitors for therapeutic purposes. Our results suggest also that the true behavior of other inhibitors may have been masked when assayed with only p-nitrophenylphosphate as substrate.

Keywords: Bis(acetylacetonato)oxidovanadium(IV); Protein tyrosine phosphatase-1B; Steady-state kinetics; Uncompetitive inhibition; VO(acac)2; Vanadyl (VO2+) chelates.

Fig. 1

Comparison of chemical bonding structures of (a) bis(acetylacetonato)oxidovanadium(IV), VO(acac)₂; (b) bis(2-ethyl-maltolato)oxidovanadium(IV), VO(Et-malto)₂; and (c) bis(3-hydroxy-2-methyl-4(1H)pyridinonato)oxidovanadium(IV), VO(mpp)₂.

Fig. 2

Comparison of UV absorption spectra of VO(acac)₂ in the absence (Panel A) and presence (Panel B) of PTP1B. In Panel A an aliquot of VO(acac)₂ dissolved in neat DMSO was added to a final concentration of 6 × 10⁻⁵ M and mixed in N₂-purged 0.1 M NaCl buffered to pH 5 with 0.01 M sodium acetate at 22° C. Spectra were collected, in direction of the arrow, at 0, 1.5, 3, 4.5, 6, and 9 min. In Panel B an aliquot of VO(acac)₂ was added and allowed to come to equilibrium as in Panel A. Small aliquots of a stock PTP1B solution were then added to final concentration of 0, 0.089, 0.18, 0.23, 0.35, 0.43, 0.51, and 0.59 ×10⁻⁶ M, as indicated by the arrow, and mixed. The spectra were collected only after equilibrium had been reached for addition of each enzyme aliquot. The addition of enzyme aliquots contributed to less than a 2% increase in the total volume of the solution in the cuvette.

Fig. 3

Comparison of UV absorption spectra of VO(Et-maltol)₂ in the absence (Panel A) and presence (Panel B) of PTP1B. In Panel A an aliquot of VO(Et-maltol)₂ dissolved in neat DMSO was added to a final concentration of 4.5 × 10⁻⁵ M to a solution and mixed in N₂-purged 0.1 M NaCl buffered to pH 5 with 0.01 M sodium acetate at 22° C. In Panel B an aliquot of VO(Et-maltol)₂ was added to a concentration of 3 × 10⁻⁵ M in acetate buffered 0.1 M NaCl as in Fig. 2B. Aliquots of a stock PTP1B solution were added, in the order indicated by the arrow, to final concentrations of 1.0, 3.0, 3.9, and 4.9 × 10⁻⁷ M, respectively, and mixed. The spectra were collected only after equilibrium had been reached for addition of each enzyme aliquot. The two different concentrations of the VO²⁺-chelate in Panels A and B were chosen to maintain approximately equivalent signal-to-noise in both panels for comparison. Other conditions as in Fig. 2.

Fig. 4

Lineweaver-Burk plots of initial velocity data of PTP1B catalyzed hydrolysis of pNPP in the presence of VO(acac)₂. Lines are plotted according to parameters obtained from hyperbolic fits to the data as described in the text for (Panel A) competitive inhibition and (Panel B) noncompetitive inhibition. The reaction mixture consisted of N₂-purged 0.1 M NaCl buffered to pH 5 with 0.01 M sodium acetate. Enzyme concentration was 3.2 × 10⁻⁸ M. Inhibitor concentrations were (× 10⁶ M): ● (0), ▲ (6.0), ▼ (10), ◆ (14). The area near the origin is expanded in the inset to emphasize that the intersection of each straight-line with the ordinate was drawn according to parameters based on hyperbolic fits to initial velocity data.

Fig. 5

Lineweaver-Burk plots of initial velocity data of PTP1B catalyzed hydrolysis of pNPP in the presence of VO(Et-malto)₂. Solution conditions and determination of kinetic parameters from hyperbolic plots as in Fig. 3. Enzyme concentration was 2.9 × 10⁻⁸ M. Inhibitor concentrations were (× 10⁶ M): ■ (0); ● (2.0), ▲ (3.0), ▼(4.0), ◆ (6.0).

Fig. 6

Lineweaver-Burk plot of inhibition of PTP1B catalyzed hydrolysis of EGFR^988-998 in the presence of VO(acac)₂. The double reciprocal plot was constructed on the basis of the parameters generated from hyperbolic fits of initial velocity data. For each inhibitor concentration, K_M and V_max values determined in the absence of the inhibitor were fixed. Competitive, noncompetitive, and uncompetitive models of inhibition were then evaluated on the basis of hyperbolic fits to the full data set by varying K_I and, for noncompetitive and uncompetitive models, V_max. The final interpretation of the mode of inhibition for the chelate was selected on the basis of the best fit, evaluated visually on the basis of double-reciprocal plots and quantitatively by the χ² value. The reaction mixture consisted of N₂-purged 0.1 M NaCl buffered to pH 6 with 0.01 M Tris-Ac. The concentration of PTP1B was 3.75 × 10⁻⁹ M. Initial velocity data in the absence of inhibitor are indicated by squares (■). VO(acac)₂ concentrations were (× 10⁹ M): ● (50), ▲ (100), ▼ (150).

Fig. 7

Lineweaver-Burk plot of initial velocity data of PTP1B catalyzed hydrolysis of EGFR^988-998 in the presence of VO(Et-malto)₂. Conditions as in Fig. 3. Initial velocity data in the absence of inhibitor are indicated by squares (■). VO(Et-malto)₂ concentrations were (× 10⁹ M): ▼ (30), ● (60), ▲ (90).

Scheme 1