Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Abstract

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K(m)) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-A resolution (final R(free) value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to lambda protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Fig. 1.

Effect of leaving group pK_a on k_cat (A) and k_cat/K_m (B) of sweet potato (•), red kidney bean (○), and pig PAP (▴). The substrates and their corresponding leaving group pK_a values are listed in Table 3. The lines were drawn using linear regression.

Fig. 2.

Stereodiagram of the active-site residues in sweet potato PAP. Side chains that coordinate directly to the metal ions are shown as stick models. Side chains that form the active site are shown as ball-and-stick models. Carbon atoms colored gray represent side chains that are conserved between red kidney bean and sweet potato PAP. Carbon atoms colored yellow are nonconserved, and the name of the equivalent residue in red kidney bean PAP is in brackets. Primed amino acid residues are from the neighboring subunit.

Fig. 3.

Stereodiagram of the binuclear metal center in the sweet potato PAP–phosphate complex. Overlayed in blue is the difference electron density, contoured at 3.0 σ, before phosphate was built into the model. Also shown are the phosphate (yellow sticks) and the μ–oxo (yellow ball) of pig PAP after superimposition of the metal ions in sweet potato and pig PAP. It was not possible to fit the phosphate in an equivalent conformation as observed in pig PAP.

Fig. 4.

The role of H295 and E365 in the orientation of the substrate and the stabilization of the transition state. At low pH, E365 acts as proton donor for the leaving group; at higher pH, H295 occupies this role. The oxygen atom located between the two metal ions originates from the bridging oxygen, the proposed nucleophile (see Figs. 6 and 7).