The reaction mechanism of the Ga(III)Zn(II) derivative of uteroferrin and corresponding biomimetics

Abstract

Purple acid phosphatase from pig uterine fluid (uteroferrin), a representative of the diverse family of binuclear metallohydrolases, requires a heterovalent Fe(III)Fe(II) center for catalytic activity. The active-site structure and reaction mechanism of this enzyme were probed with a combination of methods including metal ion replacement and biomimetic studies. Specifically, the asymmetric ligand 2-bis{[(2-pyridylmethyl)-aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol and two symmetric analogues that contain the softer and harder sites of the asymmetric unit were employed to assess the site selectivity of the trivalent and divalent metal ions using (71)Ga NMR, mass spectrometry and X-ray crystallography. An exclusive preference of the harder site of the asymmetric ligand for the trivalent metal ion was observed. Comparison of the reactivities of the biomimetics with Ga(III)Zn(II) and Fe(III)Zn(II) centers indicates a higher turnover for the former, suggesting that the M(III)-bound hydroxide acts as the reaction-initiating nucleophile. Catalytically active Ga(III)Zn(II) and Fe(III)Zn(II) derivatives were also generated in the active site of uteroferrin. As in the case of the biomimetics, the Ga(III) derivative has increased reactivity, and a comparison of the pH dependence of the catalytic parameters of native uteroferrin and its metal ion derivatives supports a flexible mechanistic strategy whereby both the mu-(hydr)oxide and the terminal M(III)-bound hydroxide can act as nucleophiles, depending on the metal ion composition, the geometry of the second coordination sphere and the substrate.

Metal-Ion Mutagenesis: Conversion of a Purple Acid Phosphatase from Sweet Potato to a Neutral Phosphatase with the Formation of an Unprecedented Catalytically Competent MnIIMnII Active Site

“…The data points are determined experimentally and the solid lines represent fits to the data using eqs 1 and 2 and procedures described elsewhere. 22 Figure 5.…”

“…The pH dependence of the k cat and k cat /K M values were fitted to eqs 1 and 2 as described elsewhere. 22,33 where K esn (n ) 1, 2, 3) represents a protonation equilibrium of the enzyme-substrate complex and k catn (n ) 1, 2, 3, 4) corresponds to the activity of the associated protonation states. …”

“…16,17 In fact, this approach has been successfully employed to generate metalloderivatives of PAPs. [16][17][18][19][20][21][22][23] For instance, the Fe II in pig PAP (uteroferrin) can be replaced by Zn II without a significant change in activity, whereas the Mn II , Ni II , Cu II , and Co II derivatives display 10-50% activity of the native enzyme. 19,[21][22][23] Although the divalent ion can be replaced, there are no reports of catalytically active dinuclear homodivalent PAPs.…”

GaIII Complexes as Models for the MIII Site of Purple Acid Phosphatase: Ligand Effects on the Hydrolytic Reactivity Toward Bis(2,4-dinitrophenyl) phosphate

“…3 Evidence has been obtained that uteroferrin PAP (Fe III Fe II core) hydrolyzes phosphate diesters by the same mechanism proposed for kbPAP (Scheme 1a), while sweet potato PAP (Fe III Mn II core) and the non-native Ga III Fe II , Fe III Ni II and Ga III Zn II forms of uteroferrin PAP employ a catalytic mechanism involving bridging substrate coordination and nucleophilic attack by a bridging hydroxo or oxo group (Scheme 1b). 3,14 Scheme 1. Proposed mechanisms for purple acid phosphatase catalyzed phosphate ester hydrolysis.…”

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

“…Although it is believed that PAPs are only active in the heterovalent state [16], phosphatase activity has been reported for a number of diferric model complexes [17][18][19][20][21][22][23] [8,9,[17][18][19][20][21][22][23][24][25], particularly those for which more than two deprotonation equilibria are considered as part of the profile [9,13,14].…”