Promiscuous Sulfatase Activity and Thio-Effects in a Phosphodiesterase of the Alkaline Phosphatase Superfamily<xref ref-type="fn" rid="fn1"></xref>

The nucleotide phosphodiesterase/pyrophosphatase from Xanthomonas axonopodis (NPP) is a structural and evolutionary relative of alkaline phosphatase that preferentially hydrolyzes phosphate diesters. With the goal of understanding how these two enzymes with nearly identical Zn2+ bimetallo sites achieve high selectivity for hydrolysis of either phosphate monoesters or diesters, we have measured a promiscuous sulfatase activity in NPP. Sulfate esters are nearly isosteric with phosphate esters but carry less charge, offering a probe of electrostatic contributions to selectivity. NPP exhibits sulfatase activity with kcat/KM value of 2 × 10-5 M-1 s-1, similar to the R166S mutant of alkaline phosphatase. We further report the effects of thio-substitution on phosphate monoester and diester reactions. Reactivities with these noncognate substrates illustrate a reduced dependence of NPP reactivity on the charge of the nonbridging oxygen situated between the Zn2+ ions relative to that in alkaline phosphatase. This reduced charge dependence can explain about 102 of the 107-fold differential catalytic proficiency for the most similar monoester and diester substrates in the two enzymes. The results further suggest that active site contacts to substrate oxygen atoms that do not contact the Zn2+ ions may play an important role in defining the selectivity of the enzymes. The biological reactions of phosphate esters drive energy flux and genetic inheritance for all living things (1), and enzymes that catalyze reactions at phosphorus have some of the largest rate accelerations known (2). A significant challenge in understanding the chemistry of biological phosphoryl transfer is in defining the mechanisms through which phosphoryl transfer enzymes achieve their tremendous chemical selectivity. Alkaline phosphatase from Escherichia coli (AP) catalyzes the hydrolysis of phosphate monoesters using two active site Zn2+ ions and is the most extensively studied member of the alkaline phosphatase superfamily of enzymes (3-5). Nucleotide phosphodiesterase/pyrophosphatases are members of the alkaline phosphatase superfamily (5, 6) that preferentially hydrolyze phosphate diesters and are believed to play roles in diverse processes including bone mineralization, insulin signaling, and cell migration (7, 8). Remarkably, the differential catalytic specificity between AP and Xanthomonas axonopodis nucleotide phosphodiesterase/pyrophosphatase (NPP) spans 15 orders of magnitude, yet in crystal structures, the active site zinc ions and their six side chain ligands are identical and overlay with 0.3 Å rmsd (9). Because AP and NPP are structurally similar but highly specific for either phosphate monoester or diester hydrolysis, these enzymes offer an opportunity to learn how structural features modify and extend enzymatic function beyond the basic two-metal-ion mechanism (10, 11). A prior study proposed that the strong selectivity of AP for phosphate monoesters might arise from the interaction of negative charge on a nonbridging oxygen atom with the Zn2+ ions (Figure 1), consistent with the perspective that the bimetallo site is central to catalysis (12). In that study, a series of substrates of varying nonbridging oxygen charge was tested in the R166S variant of AP, and a steep linear correlation with reactivity was observed, with 31 kcal/mol of transition state stabilization per unit charge. The study suggested that AP might achieve a substantial degree of selectivity for hydrolysis of phosphate monoesters over phosphate diesters and sulfate esters because of the increased negative charge on the nonbridging oxygens of phosphomonoesters relative to diesters and sulfate esters. While the interaction of Zn2+ ions with nonbridging oxygen charge provides a plausible mode of discrimination † This work was supported by a grant from the NIH to D.H. (GM64798). J.K.L. was supported by an NIH postdoctoral fellowship (F32 GM080865). * Address correspondence to D.H. at the Department of Biochemistry, Beckman Center, B400, Stanford University, Stanford, CA 943055307. Phone 650-723-9442. Fax: 650-723-6783. E-mail: herschla@ stanford.edu 1 Abbreviations: AP, Escherichia coli alkaline phosphatase; NPP, Xanthomonas axonopodis nucleotide phosphodiesterase/pyrophosphatase; pNPS, 4-nitrophenyl sulfate; pNPP, 4-nitrophenyl phosphate; pNPPS, 4-nitrophenyl phosphorothioate; bis-pNPP, bis-4-nitrophenyl phosphate; MepNPP, methyl 4-nitrophenyl phosphate; MepNPPS, methyl 4-nitrophenyl phosphorothioate. FIGURE 1: A nonbridging oxygen lies between two Zn2+ ions in AP and NPP transition state models (see reference 9 and Figure 7 for more detailed comparisons). The ester substituent of diesters is indicated by R′. Biochemistry 2008, 47, 12853–12859 12853 10.1021/bi801488c CCC: $40.75  2008 American Chemical Society Published on Web 11/01/2008 for AP, this model leaves open the question of how NPP could use the same metal site to preferentially hydrolyze lesscharged phosphate diesters. To define the relationship between nonbridging oxygen charge and reactivity in NPP, we looked for a promiscuous sulfatase activity and evaluated the effects of thio-substitution on phosphate monoester and diester substrates in NPP. Of particular importance is the reactivity of NPP toward sulfate monoesters, as they are nearly isosteric with phosphate esters (13, 14) but carry a full unit less negative charge. If NPP has a similar sensitivity toward nonbridging oxygen charge to that of AP, then the sulfatase activity of NPP would be expected to be substantially reduced relative to AP, in accord with its reduced phosphomonoesterase activity. Alternatively, if the reduced negative charge of phosphate diesters plays a significant role in their favored hydrolysis by NPP, then a corresponding enhanced sulfatase activity of NPP would be predicted. MATERIALS AND METHODS Substrates. p-Nitrophenyl phosphate (pNPP, Scheme 1) and p-nitrophenyl sulfate (pNPS) were obtained from Sigma. p-Nitrophenyl phosphorothioate (pNPPS) was synthesized previously (15) and recrystallized from anhydrous methanol. Methyl p-nitrophenyl phosphorothioate (MepNPPS) was synthesized from dimethyl chlorothiophosphate and pnitrophenol (16). The product was purified in two silica gel chromatography steps, one with 20% ethyl acetate, 0-20% methanol, and hexanes, and another with 10% triethylamine, 85% acetone, and 5% methanol. Residual solvent was removed by repeated evaporation from aqueous solution. These steps yielded pure MepNPPS oil as the triethylammonium salt: 1H NMR (D2O) δ 8.27 (d, 2H, Ph), δ 7.38 (d, 2H, Ph), δ 3.75 (d, 3H, CH3, JCH3-P ) 13.2), δ 3.19 (q, 6H, CH2, NH(CH2CH3)3), δ 1.27 (t, 9H, CH3, NH(CH2CH3)3); 31P NMR (D2O) δ 54.34. These chemical shifts are consistent with expectations from related compounds (16, 17). Mass spectrometry and reaction to completion with NPP further established identity and purity of the substrate. Protein Expression and Purification. NPP was expressed, purified, and quantified as previously described (9). Kinetic Assays of NPP-Catalyzed Reactions. For each substrate, kcat/KM values were measured under conditions where initial rates remained linear with both enzyme and substrate concentration over at least 10-fold ranges (V0) (kcat/ KM)[E][S]). Comparison of kcat/KM values is desirable as kcat/ KM reflects the free energy difference between the bound transition state and free enzyme and substrate in solution. Furthermore, saturating behavior is not observed for several substrates in NPP (9). The absorbance of 4-nitrophenolate was monitored continuously at 400 nm [ε) 16652 M-1 cm-1 (pH 8.0); ε ) 4382 M-1 cm-1 (pH 6.5)] in quartz cuvettes. For pNPS activity, absorbance at 500 nm (where 4-nitrophenolate does not absorb) was subtracted from each reading to minimize noise due to cuvette movement. Because pNPS activity was monitored continuously over time periods of up to 48 h, full activity of enzyme in the cuvettes was verified following all kinetic runs by removing aliquots directly from the cuvettes and assaying in separate reactions with MepNPP. All measurements other than with pNPS were taken in 0.1 M Tris pH 8.0, 0.5 M NaCl, and 100 μM ZnCl2 at 25 °C. Sulfatase activity was measured at lower pH (25 °C, 0.1 M MES, pH 6.5, 0.5 M NaCl, 100 μM ZnCl2) to permit higher enzyme concentration without precipitation. NPP exhibits a flat pH-rate profile within this region (9), and rates that could be measured at pH 8 were within error of those obtained at pH 6.5. For the MepNPPS substrate, reaction to completion and subsequent 31P and 1H NMR analysis indicated that only one enantiomer reacts efficiently. Thus, for determination of MepNPPS activity, values for substrate concentrations reflected only the reactive enantiomer. Inhibition of NPP Enzymatic ActiVity with AMP. Inhibition with AMP was measured for pNPPS and MepNPPS activity with NPP under subsaturating conditions as described for kinetic assays. Values of Ki were obtained by nonlinear fitting of the data to the Michaelis-Menten equation with competitive inhibition. Because of the low activity of NPP toward pNPS, protein concentrations of 10-100 μM were used in kinetic assays. As a result, the simplifying assumption that [I]free ) [I]total does not hold (because [E] is similar to or greater than Ki), and the commonly used form of the Michaelis-Menten equation with competitive inhibition could not be used. Instead, the concentration of free enzyme was calculated by solving for the free enzyme concentration from the expression of the inhibition equilibrium. The quadratic equation was used to obtain an expression for free enzyme concentration using values of [I]total, Ki, and [E]total, and this was substituted into the equation for initial rates under subsaturating conditions. Expected initial rates were calculated as follows: