Calcineurin redox implications for catalysis

400 mM ascorbate, lOOmM yS-mercaptoethanol, 50 mM dithiothreitol, etc.) and 0.02-6mM Fe(NH4)2(S04)2). Under these conditions, the metal cluster of purple acid phosphatase becomes poised in the active Fe -Fe oxidation state [41,46, 51-53]. If calcineurin were to contain a dinuclear iron cluster, the protective effects of ascorbate and the ability to reactivate calcineurin in the presence of Fe(NH4)2(S04)2 would similarly stabilize the metal cluster in the Fe -Fe oxidation state. Careful spectroscopic and mechanistic studies will be required in order to determine whether this is the case. In addition, studies which show a correlation between intracellular redox state and the activity of calcineurin are being pursued in order to provide insight into the regulation of calcineurin in vivo. 

The dependence of the activity of calcineurin on the redox state of the metal center highlights its importance for catalysis and provides clues to its function in that process. Site-directed mutagenesis studies of PPl, calcineurin, and bacteriophage X protein phosphatase have also provided insights regarding the roles of non-ligand active site residues. Furthermore, the contributions afforded by studies of synthetic model compounds which mimic features of metallophosphatase active sites provide important clues to possible catalytic mechanisms. Indeed many of these models exhibit impressive rate enhancements for phosphate and phosphonate ester hydrolysis [54-62]. In this section we discuss current models regarding the mechanism of phosphate ester hydrolysis by calcineurin and other metallophosphatases in consideration of these studies. 

Similar to the metallophosphatases, alkaline phosphatase is a metalloenzyme containing two Zn + ions and a Mg ion as active site cofactors. Despite the similarity with regard to an active site metal cluster, most of the data support a direct 

In the metallophosphatases, there is evidence for a metal coordinated hydroxide serving as the nucleophile in the reaction. The enzymatic rate for purple acid phosphatase exhibits bell-shaped kinetics with respect to pH and a requirement for a group which needs to be deprotonated with a pA of 4.5 [53], Assignment of this group to a metal-coordinated water molecule is consistent with pH titrations of the EPR signal of the Fe +-Fe + cluster which demonstrated the presence of an ioniz- 

A nucleophilic hydroxide coordinated to one metal ion (terminal coordination) or bridged to both metals (, -hydroxo coordination) is possible both types are present in the X-ray structures. We first consider the possibility that the nucleophile in the reaction is a water molecule coordinated to the Fe + ion of the binuclear metal center. A terminal metal-hydroxo species serving as a nucleophile in an hydrolysis reaction has precedence in model chemistry [62]. The Lewis acidity of the metal, which plays a role by decreasing the pXa of the coordinated water molecule, is more favorable for Fe + than Fe +. The pAaS of water coordinated to aqueous Fe and Fe + ate 2.13 and 8.44, respectively, a 10 -fold difference in acidity [74]. A lower pAa makes it easier to deprotonate to form the hydroxide, the putative nucleophile in the reaction. The loss of activity of Fe -Fe calcineurin upon reduction to the Fe -Fe + state may therefore reflect the poorer Lewis acidity of Fe + and the requirement for an hydroxide coordinated to the Fe ion in the Ml site [35]. 

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