The structure of inositol monophosphatase has been determined to 2.60 A resolution in complexes with Mn2+ and with Mn2+ and phosphate. In the Mn2+ complex, three metal cations and one Cl were bound in the active site on each of the two subunits of the enzyme. Ligands to the three metals include the side chains of Glu 70, Asp 90, Asp 93, and Asp 220, t he carbonyl group of Ile 92, several solvent molecules and the chloride, which is a ligand to each of the cations. When phosphate is soaked into these Mn2+ cocrystals, one of the three Mn2+ ions is expelled from the active site, leaving metal ions with octahedral and tetrahedral coordination geometry. In addition, the structure of apoinositol monophosphatase was determined to 2.5 A resolution. Residues 70-75, a two-turn helical segment which is involved in metal coordination, moves away from the metal binding site by 2-3 A in the absence of cations. Residues 30-40, which wrap around the metal binding site and interact with the metal indirectly through solvent molecules and protein ligands to the metal, become disordered in the absence of metal. In various metal complexes, segmental mobility is also observed in the residues which form the metal binding sites. The results of these studies of the interaction of inositol monophosphatase with cations suggest that the enzyme accomplishes phosphate ester hydrolysis using two metal ions, one with octahedral and one with tetrahedral coordination geometry. Broad metal-binding specificity appears to result from extensive flexibility in several of the protein segments which contribute metal ligands, from the presence of alternate metal ligands and from metal coordination spheres which include water molecules.

The structures of ternary complexes of human inositol monophosphatase with inhibitory Gd3+ and either D- or L-myo-inositol 1-phosphate have been determined to 2.2-2.3 A resolution using X-ray crystallography. Substrate and metal are bound identically in each active site of the phosphatase dimer. The substrate is present at full occupancy, while the metal is present at only 35% occupancy, suggesting that Li+ from the crystallization solvent partially replaces Gd3+ upon substrate binding. The phosphate groups of both substrates interact with the phosphatase in the same manner with one phosphate oxygen bound to the octahedrally coordinated active site metal and another oxygen forming hydrogen bonds with the amide groups of residues 94 and 95. The active site orientations of the inositol rings of D- and L-myo-inositol 1-phosphate differ by rotation of nearly 60 degrees about the phosphate ester bond. Each substrate utilizes the same key residues (Asp 93, Ala 196, Glu 213, and Asp 220) to form the same number of hydrogen bonds with the enzyme. Mutagenesis experiments confirm the interaction of Glu 213 with the inositol ring and suggest that interactions with Ser 165 may develop during the transition state. The structural data suggest that the active site nucleophile is a metal-bound water that is activated by interaction with Glu 70 and Thr 95. Expulsion of the ester oxygen appears to be promoted by three aspartate residues acting together (90, 93, and 220), either to donate a proton to the leaving group or to form another metal binding site from which a second Mg2+ coordinates the leaving group during the transition state.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a {-associated protein of Mr 70,000 as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) Many of the details describing the biochemical cascade responsible for T-cell receptor (TCR) activation and development have recently been elucidated [for a detailed review, see Chan et al. (1)]. The most widely accepted model includes data from studies with chimeric molecules and reconstituted receptors (2-4) which supports engagement of the TCR followed by phosphorylation of the immune-receptor tyrosine activating motifs (ITAMs) in the cytoplasmic domains of the CD3 and ; chains by src-family protein tyrosine kinases (PTKs) (3,5). This phosphorylation in turn is followed by recruitment of a c-associated protein of Mr 70, Recent evidence, the most compelling of which comes from human genetic studies (10-14), reports patients with severe combined immunodeficiency due to Zap-70 deficiency. These data immediately implicated Zap-70 as a critical enzyme in T-cell function and development and heightened interest in determining the biochemical and enzymatic role Zap-70 plays in these processes.The current study was designed to investigate the mechanism of activation for Zap-70 catalysis by utilizing purified components of the TCR signaling pathway, including monomeric and dimeric forms of the cytoplasmic C chain (as determined by mass spectrometry and size exclusion chromatography), bisphosphorylated C peptides, and Flag epitopetagged Zap-70. Capture of 33P-labeled product on phosphocellulose paper and autoradiography were used to monitor product formation. To date, most previous published reports on the activation of Syk-family PTKs (i.e., Syk and Zap-70) describe studies utilizing immunoprecipitates of cell lysates or permeabilized cells (15)(16)(17)(18)(19).The major finding of our study is that the catalytic activity of purified Flag-Zap-70 with regard to autophosphorylation and phosphorylation of an exogenous substrate, gastrin117, is increased -5-and 20-fold, respectively, in the presence of 125 nM phosphorylated glutathione S-transferase (GST)-Zeta or GST-Zeta-1 cytoplasmic domain, but not by various forms of monomeric ; chain and bisphosphorylated C peptides. Taken together our data suggest an activation mechanism for Zap-70 catalysis which occurs by an intermolecular trans-phosphorylation reaction. MATERIALS AND METHODSExpression of Flag-Zap-70. Human Zap-70 was expressed as a fusion with the Flag epitope at its N terminus (residues MDYKDDDDKH) in baculovirus infected Sf9 cells. Infected cells were harvested after 48 hr and cell lysates were prepared.Flag-Zap-70 was purified from Sf9 lysates by affinity chromatography utilizing anti-Flag M2 affinity gel (Kodak). A final concentration of 500 mM Na...

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