Kinetic and Spectroscopic Analyses of Mutants of a Conserved Histidine in the Metallophosphatases Calcineurin and λ Protein Phosphatase

Abstract

Calcineurin belongs to a family of serine/threonine protein phosphatases that contain active site dinuclear metal cofactors. Bacteriophage protein phosphatase is also considered to be a member of this family based on sequence comparisons (Lohse, D. L., Denu, J. M., and Dixon, J. E. (1995) Structure 3, 987-990). Using EPR spectroscopy, we demonstrate that protein phosphatase accommodates a dinuclear metal center. Calcineurin and protein phosphatase likewise contain a conserved histidine that is not a metal ligand but is within 5 Å of either metal in calcineurin. In this study the conserved histidine in calcineurin was mutated to glutamine and the mutant protein analyzed by EPR spectroscopy and kinetic methods. Parallel studies with an analogous protein phosphatase mutant were also carried out. Kinetic studies using paranitrophenyl phosphate as substrate showed a decrease in k cat of 460-and 590-fold for the calcineurin and protein phosphatase mutants, respectively, compared with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease in k cat of 1,300-fold for calcineurin. With the analogous protein phosphatase mutant, k cat decreased 530-fold compared with wild type protein phosphatase using phenyl phosphate as a substrate. EPR studies of the iron-reconstituted enzymes indicated that although both mutant enzymes can accommodate a dinuclear metal center, spectroscopic differences compared with wild type proteins suggest a perturbation of the ligand environment, possibly by disruption of a hydrogen bond between the histidine and a metal-coordinated solvent molecule.Calcineurin, also known as protein phosphatase 2B, consists of a 58-kDa catalytic subunit, calcineurin A, and a 19-kDa regulatory subunit, calcineurin B. It is a serine/threonine protein phosphatase whose activity is regulated by Ca 2ϩ /calmodulin. Calcineurin is the target of the immunosuppressant drugs cyclosporin A and FK506 (1, 2). These drugs bind to intracellular proteins, termed immunophilins; cyclophilin is the binding protein for cyclosporin A, and FK506 binds to the FK506-binding proteins. The complex of immunosuppressant drug and immunophilin in turn binds to and inhibits the phosphatase activity of calcineurin. Calcineurin inhibition prevents the transcriptional activation of the interleukin 2 gene in helper T cells, leading to suppression of the immune response.Calcineurin is a member of the class of serine/threonine protein phosphatases, whose members include protein phosphatases 1 (PP1) 1 and 2A (PP2A), phosphatases essential for a number of signal transduction pathways in eukaryotic cells (3, 4). Another protein phosphatase from bacteriophage , PP, also belongs to this family (5). In addition, a number of less characterized enzymes containing the "phosphoesterase" consensus motif of this family, DXH(X) n GDXXD(X) n GNHD/E, have been identified via protein sequence comparisons (6, 7). It has been hypothesized that this motif provides a scaffold for an active site dinuclear meta...

Redox Control of Calcineurin by Targeting the Binuclear Fe2+-Zn2+ Center at the Enzyme Active Site

“…Klee and co-workers (6) suggest the existence of superoxide-sensitive ferrous iron, but on the basis of spectroscopic and kinetic studies on isolated bovine CaN it was later suggested by Rusnak and co-workers (14,15) that native CaN exists in a redoxinsensitive Fe 3ϩ -Zn 2ϩ form. A redox-sensitive Fe 3ϩ -Fe 2ϩ form of CaN resembling the well characterized binuclear center in bovine spleen purple acid phosphatase was artificially obtained by metal exchange (14,16). It was hypothesized that this form could also contribute to redox sensitivity of native CaN in tissue homogenates (9,17).…”

Design, synthesis and testing of purple acid phosphatase inhibitors for the development of anti-osteoporotic drugs

“…PAPs' main function is protein dephosphorylation, which is the main mechanism in cell signal transduction pathways [72][73]75 and hence important cellular processes in general. Thus, PAPs have many significant biological functions.…”

“…This is in agreement with previous studies. [72][73] Figure 23: SDS-PAGE analysis of the fractions collected from the chromatography of allantoic fluid on a gel filtration, Sephacryl S200 column, stained using Bio-Safe Coomassie G-250. The fractions contain pure pPAP as shown by the single band for each fraction (in the box).…”

“…81 The overall catalytic step involves a direct transfer of the phosphate moiety of the substrate to a nucleophilic solvent molecule that is coordinated to a metal ion. 55,65,[72][73][76][77]107 The first step involves the binding of the substrate to PAP to form a pre-catalytic complex. There is no experimental data of this structure currently available, however the crystal structure of red kidney bean PAP (rkbPAP)-sulfate complex solved by Schenk and co-workers at 2.4 Å (Figure 6) shows that instead of binding directly to the metal ions, the sulfate is positioned in the second coordination sphere, and is stabilised via hydrogen bonds with the µ-hydroxide, His202, His295, His296 and Asn201 (residue numbers refer to the sequence of rkbPAP).…”

“…Upon treatment with β-mercaptoethanol (BME) and ferrous ammonium sulfate, the pink form of pPAP had an absorption maximum at 510 nm, with a marked decrease in the 320 nm shoulder, consistent with that in the literature. 55,[71][72][73] Pig PAP was reduced to convert it into its active, pink form (Fe(III)Fe(II)) by incubating it with BME and ferrous ammonium sulfate for ten minutes at 37 °C to be used in the kinetic assays.…”