Inhibition of Bacteriophage λ Protein Phosphatase by Organic and Oxoanion Inhibitors

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Abstract
Bacteriophage λ protein phosphatase (λPP) with Mn2+ as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of λPP, albeit with inhibition constants (Ki) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (Ki = 5.1 ± 1.6 μM) whereas phosphonoacetic acid (Ki = 380 ± 45 μM) and acetohydroxamic acid (Ki > 75 mM) modestly inhibited λPP. Low-temperature EPR spectra of Mn2+-reconstituted λPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn2+ ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn2+)2 dimer is most prominent upon the addition of orthovanadate (Ki = 0.70 ± 0.20 μM) and PhAH, indicating that these inhibitors tightly interact with the (Mn2+)2 form of λPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by λPP and other serine/threonine protein phosphatases.