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Fig. 5. Overall fold of sweet potato purple acid phosphatase (PAP). The polypeptide is colored from blue (N-terminal end of subunit A) to red (C-terminal end of subunit B). The bound phosphate is depicted as a ball-and-stick and the metal ions as spheres (dark red, Fe; silver, Mn). Sweet potato PAP consists of two identical polypeptides of 435-aa residues (1). The asymmetric unit consists of 2 polypeptide chains, 190 water molecules, 21 carbohydrate molecules, 2 phosphate molecules, 2 Fe, and 2 Mn nuclei. One subunit (A) shows continuous electron density from the N-terminal end to W424, and the other subunit (B) shows continuous electron density from the N-terminal end to S426. Amino acids L1, N62, S63, and K64 in both subunits and K67 in subunit B do not show any density for the side chains and therefore were modeled as Ala. The two polypeptides in the asymmetric unit are structurally indistinguishable; the only region where there is significant variation from this value is between amino acids S63 and A68, where the temperature factors reach a maximum of 80 Ų. This segment of polypeptide is at the bottom of the N-terminal domain. The metal ions are well resolved in both subunits (see text). The low temperature factors for the Mn ions imply a high occupancy for these sites. Metal analysis of the samples used in crystallization indicate 1 atom of iron and » 0.75 atoms of Mn per subunit (1, 2). The overall structures of red kidney bean (3) and sweet potato PAP are highly conserved. A superposition of 411 Ca atoms of the relevant polypeptides results in an average rms deviation (rmsd) value of 0.85 Å, whereas the 822 Ca atoms in the dimers can be aligned with an overall rmsd of 1.02 Å. The small difference in rmsd values between monomer and dimer superpositions indicate that the red kidney bean and sweet potato enzymes associate in an identical manner. There are several regions where the structures are substantially different. One is at E365, close to the active site (see text). The other is between residues Y48 and V56. The only insertion or deletion of sequence occurs here; Y49 is an insertion in the sweet potato PAP sequence relative to red kidney bean PAP. This structural difference is unlikely to have any impact on the catalytic activity of the two enzymes. However, the role of the N-terminal domain is still unknown, and it is possible that structural variations in this domain may be important in interactions between PAP and its potential targets. In this respect, we note that in mammalian PAPs, a region remote from the catalytic site has been identified that interacts with an osteoblast-specific protein (TRIP-1). It is believed that this protein-protein interaction is crucial for osteoblast differentiation (4).

1. Schenk, G. S., Ge, Y., Carrington, L. E., Wynne, C. J., Searle, I. R., Carroll, B. J., Hamilton, S. & de Jersey, J. (1999) Arch. Biochem. Biophys. 370, 183–189.

2. Schenk, G., Carrington, L. E., Hamilton, S. E., de Jersey, J. & Guddat, L.W. (1999) Acta Crystallogr. D 55, 2051–2052.

3. Sträter, N., Klabunde, T., Tucker, P., Witzel, H. & Krebs, B. (1995) Science 268, 1489–1492.

4. Sheu, T. J., Schwarz, E. M., Martinez, D. A., O’Keefe, R. J., Rosier, R. N., Zuscik, M. J. & Puzas, J. E. (2003) J. Biol. Chem. 278, 438–443.

Fig. 6. H-bond network in the active site of sweet potato PAP at pH of » 4. The H-bond pattern varies in the two subunits, indicating structural flexibility in the active site. The protonation state of phosphate is H₂PO₄^–, similar to that reported for the binuclear, Ni(II)-dependent enzyme urease (1), although the precise location of the proton in the H-bond between E365 and phosphate remains to be established. Note that the H-bond pattern indicates that O₃ is protonated. Because O₃ is proposed to originate from the metal-bridging nucleophile (see text), it follows that at pH 4.0 a m –hydroxy bridge is present. The pK_a value for the deprotonation of this hydroxide is expected to be » 4.5 because electron paramagnetic resonance spectroscopy and multifield saturation magnetization measurements indicate that at pH 4.90 a m –oxo species is present in resting sweet potato PAP (2).

1. Benini, S., Rypniewski, W. R., Wilson, K. S., Ciurli, S. & Mangani, S. (2001) J. Biol. Inorg. Chem. 6, 778–790.

2. Schenk, G., Boutchard, C. L., Carrington, L. E., Noble, C. J., Moubaraki, B., Murray, K. S., de Jersey, J., Hanson, G. R. & Hamilton, S. (2001) J. Biol. Chem. 276, 19084–19088.

Fig. 7. Proposed reaction mechanism for sweet potato PAP at low pH. After initial binding to the divalent metal ion the substrate is reoriented in the active site to form a bidentate complex. This reaction appears to be assisted by the presence of H295 and E365 from subunit A and Y258 from subunit B. The substrate is now in an ideal position for nucleophilic attack by the m –hydroxo group. Protonation of the leaving group by E365 results in the release of ROH. Subsequently, E365 forms a H-bond with phosphate (O₁ or O₄ in subunit A or B, respectively; see Fig. 6), which is bound to the active site in an unusual tridentate coordination. Exchange of the phosphate group by two water molecules regenerates the active site. Note that in this proposed mechanism only two H₂O are required to reconstitute the resting state. As a result, the Fe(III) site remains five-coordinate, consistent with the results of an electron-nuclear double resonance (ENDOR) study on pig PAP (1). At higher pH values, the protonation states of the substrate, phosphate product, and nucleophile (see Fig. 6) may change; however, the basic mechanism remains the same. The only variations are that (i) H295 acts as proton donor for the leaving ROH group, and (ii) deprotonated E365 does not form H-bonds with bound phosphate. The latter point is supported by the observation that the K_i for the competitive inhibition of sweet potato PAP by phosphate is relatively insensitive to pH changes in the range between pH 3.5 and 5 (see text). However, a marked increase in the K_i is observed at pH 7.

1. Smoukov, S. K., Quaroni, L., Wang, X., Doan, P. E., Hoffman, B. M. & Que, L., Jr. (2002) J. Am. Chem. Soc. 124, 2595–2603.