Analysis of the stoichiometric metal activation of methionine aminopeptidase

Abstract

Background: Methionine aminopeptidase (MetAP) is a ubiquitous enzyme required for cell survival and an attractive target for antibacterial and anticancer drug development. The number of a divalent metal required for catalysis is under intense debate. E. coli MetAP was shown to be fully active with one equivalent of metal by graphical analysis, but it was inferred to require at least two metals by a Hill equation model. Herein, we report a mathematical model and detailed analysis of the stoichiometric activation of MetAP by metal cofactors.

Results: Because of diverging results with significant implications in drug discovery, the experimental titration curve for Co2+ activating MetAP was analyzed by fitting with a multiple independent binding sites (MIBS) model, and the quality of the fitting was compared to that of the Hill equation. The fitting by the MIBS model was clearly superior and indicated that complete activity is observed at a one metal to one protein ratio. The shape of the titration curve was also examined for activation of metalloenzymes in general by one or two metals.

Conclusions: Considering different scenarios of MetAP activation by one or two metal ions, it is concluded that E. coli MetAP is fully active as a monometalated enzyme. Our approach can be of value in proper determination of the number of cations needed for catalysis by metalloenzymes.

Figure 1

Three different scenarios in metal activation of a metalloenzyme. The enzyme is activated by only a single metal per protein (Case 1) or by two metals (Cases 2 and 3). In each of the three cases, vertical positions represent the relative values of protein concentration, and K_D and K_D2 for the first and second metal sites M1 and M2. (A) Simulated stoichiometric titrations for Case 1. The K_D value was set to 0.2 μM with n = 1, and the protein concentration was set at 20, 2 or 0.2 μM (yielding 100:1, 10:1 or 1:1 [protein]/K_D ratio). (B) Simulated stoichiometric titrations for Case 2. Both the K_D and K_D2 values were fixed at 0.2 μM, with the total protein concentration of 20, 2 or 0.2 μM (resulting in 100:1, 10:1 or 1:1 [protein]/K_D ratio) and n = 2. (C) Simulated stoichiometric titrations for Case 3. The K_D value for the tight metal site M1 was fixed at 0.2 μM, and the K_D2 value for the weaker site was set at 20 μM, with the total protein concentration of 2000, 200 or 20 μM, giving a 100:1, 10:1 or 1:1 [protein]/K_D2 ratio.

Figure 2

Curve fitting of the experimental data of activation of E. coli MetAP by Co²⁺. (A) E. coli MetAP (20 μM) was titrated with increasing concentrations of Co²⁺, and its activity was monitored by fluorescence. This titration curve is adapted from [14]. The linear segment (diagonal dash line) corresponding to data extrapolation intercepts the maximal activity (horizontal dash line). The endpoint for the titration indicates clearly a 1:1 metal/protein molar ratio (vertical dash line). (B) The titration data in (A) was fitted with Eq. (4) (the MIBS model), giving n = 1.0 and K_D = 0.9 μM. (C) The same data was fitted with Eq. (7) (the Hill equation), giving n = 2.3 and K_0.5 = 12.1 μM. In both (B) and (C), residuals of the fitting are shown on top of the plots, and fittings at points of low metal concentrations are shown as inserts. (D) Computed fit values using both models [values for solid lines in (B) and (C)] were plotted against the raw data in a log-scale format (the MIBS model, open circles; the Hill equation model, filled circles). For a perfect fit, the plot would result in a straight line with a slope value of 1 (solid line), which is obtained by plotting the raw data against itself.