Are metal ions present at the catalytic centre?

Metal ions are involved in the catalytic mechanisms of a number of different phosphatases. Vincent and Averill have shown that the mammalian purple acid phosphatases have one and one at the active centre, while plant purple acid phosphatases have one ion and one ion. [52]. Two ions and a ion are bound at the catalytic site of E. coli alkaline phosphatase with the inorganic phosphate substrate complexed by the ions [53]. The situation with the protein serine/threonine phosphatases is less clear. Only low levels of iron and zinc (0.2 and 0.1 mol/mol protein, respectively) were found in a preparation which was probably a mixture of PP1 and PP2A [54] , whereas 0.6-0.9 mol/mol of both iron and zinc were found in a complex of PP2B catalytic and regulatory subunits [56][55] , data used to suggest that these serine/threonine protein phosphatases are metalloenzymes, with sequence similarities to the purple acid phosphatases. [57].

The property of stimulation of some forms of PP1 and PP2A and the dependence of the lambda bacteriophage phosphatase [43][42] suggests a binding site in serine/threonine protein phosphatases. However, there is no evidence that metal ions are located at the catalytic site of protein phosphatases or participate in catalysis.

Recent studies have shown that inhibitor 2 acts as a molecular chaperone in the refolding of PP1 catalytic subunit and in carrying out this function can change its dependency. Bacterially expressed PP1 alpha and PP1 beta are totally dependent on for activity. After incubation with inhibitor 2 and reactivation by phosphorylation of inhibitor 2 with glycogen synthase kinase 3 in the presence of Mg-ATP, the expressed PP1 isoforms becomes independent [58]. It was further demonstrated that PP1 could be denatured in 6M GuCl and refolded to the fully active state in the presence of 2 mM EGTA, inhibitor 2, glycogen synthase kinase 3 and Mg-ATP. Since the refolding to the fully active independent enzymes was performed in the presence of EGTA, cannot be an essential feature of the catalytic site of PP1. Instead may stimulate the expressed PP1 by maintaining the tertiary structure of the enzyme in a similar conformation to that of the native enzyme. These studies also show that if iron and zinc ions are present at the active centre, they must be bound covalently and thus it is unlikely that they would be lost during purification. They also demonstrate that although has been shown to stimulate the activity of certain forms of PP1 and PP2A [61][60][59], it is not essential for full activity and indeed there is no evidence for the presence of in the native enzyme [56][55][54].

Although the exact role of the metal binding site in serine/threonine phosphatases in either catalysis or in defining the protein conformation is unclear, there are several conserved residues that potentially could bind metal ions. Residues known to form ligands at metal ion binding sites include aspartate, asparagine, cysteine, glutamate, glutamine and histidine. The crystal structure of alkaline phosphatase shows that one ion is ligated by two His and one Asp while the second is ligated by one His and two Asps [53]. NMR studies suggests that the 2 histidines, a tyrosine and an acidic residue ligate the 2 ions bound to the catalytic site of purple acid phosphatases, [62].

Possible metal binding residues which are conserved throughout the eukaryotic protein serine/threonine phosphatase sequences are Asp 59, 66, 88 and 91; Asn 138, 324 and 331, Glu 140 (Asp in diadenosine tetra-phosphatase) and 153, and His 61, 139, 188 and 295. These residues occur in regions of the sequence predicted to form loops. When the bacteriophage and diadenosine tetra-phosphatase sequences are included only Asp 59 and 88; Asn 138; Glu 140 and His 61 and 139 are conserved.