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

Calcineurin belongs to a family of serine/threonine protein phosphatases that contain active site dinuclear metal cofactors. Bacteriophage λ 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 λ protein phosphatase accommodates a dinuclear metal center. Calcineurin and λ 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 λ protein phosphatase mutant were also carried out. Kinetic studies using paranitrophenyl phosphate as substrate showed a decrease in kcat of 460- and 590-fold for the calcineurin and λ protein phosphatase mutants, respectively, compared with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease inkcat of 1,300-fold for calcineurin. With the analogous λ protein phosphatase mutant, kcatdecreased 530-fold compared with wild type λ 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 belongs to a family of serine/threonine protein phosphatases that contain active site dinuclear metal cofactors. Bacteriophage λ 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 λ protein phosphatase accommodates a dinuclear metal center. Calcineurin and λ 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 λ protein phosphatase mutant were also carried out. Kinetic studies using paranitrophenyl phosphate as substrate showed a decrease in kcat of 460- and 590-fold for the calcineurin and λ protein phosphatase mutants, respectively, compared with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease inkcat of 1,300-fold for calcineurin. With the analogous λ protein phosphatase mutant, kcatdecreased 530-fold compared with wild type λ 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 Ca2+/calmodulin. Calcineurin is the target of the immunosuppressant drugs cyclosporin A and FK506 (1Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3546) Google Scholar, 2Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1941) Google Scholar). 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) 1The abbreviations used are: PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; λPP, λ protein phosphatase; pNPP, p-nitrophenyl phosphate; [P]-RII peptide, the phosphorylated peptide derived from the RII subunit of cyclic AMP-dependent protein kinase; λPP(H76N), λPP with asparagine in place of histidine at position 76; CN(H151Q), calcineurin with glutamine in place of histidine at position 151; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; BME, β-mercaptoethanol.1The abbreviations used are: PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; λPP, λ protein phosphatase; pNPP, p-nitrophenyl phosphate; [P]-RII peptide, the phosphorylated peptide derived from the RII subunit of cyclic AMP-dependent protein kinase; λPP(H76N), λPP with asparagine in place of histidine at position 76; CN(H151Q), calcineurin with glutamine in place of histidine at position 151; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; BME, β-mercaptoethanol. and 2A (PP2A), phosphatases essential for a number of signal transduction pathways in eukaryotic cells (3Cohen P. Cohen P.T.W. J. Biol. Chem. 1989; 264: 21435-21438Abstract Full Text PDF PubMed Google Scholar, 4Shenolikar S. Nairn A.C. Adv. Second Messenger Phosphoprotein Res. 1991; 23: 1-121PubMed Google Scholar). Another protein phosphatase from bacteriophage λ, λPP, also belongs to this family (5Zhuo S. Clemens J.C. Hakes D.J. Barford D. Dixon J.E. J. Biol. Chem. 1993; 268: 17754-17761Abstract Full Text PDF PubMed Google Scholar). In addition, a number of less characterized enzymes containing the “phosphoesterase” consensus motif of this family, DXH(X)nGDXXD(X)nGNHD/E, have been identified via protein sequence comparisons (6Koonin E.V. Prot. Sci. 1994; 3: 356-358Crossref PubMed Scopus (105) Google Scholar, 7Lohse D.L. Denu J.M. Dixon J.E. Structure. 1995; 3: 987-990Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). It has been hypothesized that this motif provides a scaffold for an active site dinuclear metal center (7Lohse D.L. Denu J.M. Dixon J.E. Structure. 1995; 3: 987-990Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Rusnak F. Yu L. Mertz P. J. Biol. Inorg. Chem. 1996; 1: 388-396Crossref Scopus (42) Google Scholar), similar to the dinuclear metal centers in PP1 (9Goldberg J. Huang H.-b. Kwon Y.-g. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (735) Google Scholar, 10Egloff M.-P. Cohen P.T.W. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (373) Google Scholar) and calcineurin (11Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (762) Google Scholar, 12Yu L. Haddy F. J. Chem. 1995; Scopus (59) Google Scholar, F. J.E. J.E. Nature. 1995; PubMed Scopus Google Scholar). A of that this in calcineurin is an center. is known the catalytic of the serine/threonine protein phosphatases, of that the dinuclear metal center is a of the active of calcineurin and PP1 that the dinuclear metal center has a ligand to that of and phosphatases, enzymes that contain either dinuclear centers that have been to be essential for catalytic activity Jr., Chem. Scopus Google Scholar, Chem. Scopus Google Scholar). studies that the of the and the both metal M.-P. Cohen P.T.W. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (373) Google Scholar, J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (762) Google Scholar, B. J. Mol. Biol. 1996; PubMed Scopus Google Scholar). of either the L. Haddy F. J. Chem. 1995; Scopus (59) Google Scholar) L., and F. in L., and F. in of calcineurin a between activity and the of the metal In to the dinuclear metal center, conserved within the active site to to of in calcineurin is histidine based on the calcineurin sequence S. K. S. K. K. E. Res. 1991; Scopus Google is not a ligand to either metal but is within 5 Å of both metal and is conserved in as PP1 and λPP (7Lohse D.L. Denu J.M. Dixon J.E. Structure. 1995; 3: 987-990Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In was to in a hydrogen bond to a metal-coordinated solvent F. J.E. J.E. Nature. 1995; PubMed Scopus Google Scholar). The of this has been by mutagenesis of both λPP and PP1 on catalytic activity protein In this study the conserved histidine in calcineurin A, was to glutamine by with calcineurin the calcineurin was to EPR spectroscopy was used to mutagenesis the ligand of the dinuclear metal center. Kinetic studies were also carried using either [P]-RII peptide as The analogous λPP mutant, was characterized by EPR in a similar and using either phenyl both mutagenesis resulted in in kcat of EPR spectroscopy of iron-reconstituted λPP that this accommodates a dinuclear metal center as for members of the EPR also that mutagenesis not of the dinuclear metal center in either calcineurin differences in the EPR of wild type mutant enzymes a perturbation of the ligand environment, possibly by disruption of a hydrogen bond between the histidine and a metal-coordinated molecule. cells were from type pNPP, phenyl and were from and were from was from was from Res. PubMed Scopus Google Scholar, M.A. PubMed Scopus Google Scholar) and to active for in and and were from and were from were from The protein used were F. Res. 1994; PubMed Scopus Google Scholar) and Haddy S. Mertz P. F. 1995; PubMed Scopus Google Scholar), the for calcineurin A and respectively, and the to 1: Scopus Google Scholar, F. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), the gene The of containing the for wild type and mutant of λPP is (5Zhuo S. Clemens J.C. Hakes D.J. Barford D. Dixon J.E. J. Biol. Chem. 1993; 268: 17754-17761Abstract Full Text PDF PubMed Google Scholar, S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The and and were from The was from The for mutagenesis was by the RII peptide and peptide were by the were by the using the with as a PubMed Scopus Google Scholar). calcineurin were by 1989; PubMed Scopus Google Scholar) using using this within with by The of calcineurin A, CN(H151Q), was using a 5 mutagenesis The to the of calcineurin A, was used for mutagenesis with the the of the mutated was phosphorylated by and used to the was from was for the by of the calcineurin A gene and the The was The of cells, with calcineurin and of the calcineurin were as F. Res. 1994; PubMed Scopus Google Scholar, Haddy S. Mertz P. F. 1995; PubMed Scopus Google Scholar). of wild type λPP was as (5Zhuo S. Clemens J.C. Hakes D.J. Barford D. Dixon J.E. J. Biol. Chem. 1993; 268: 17754-17761Abstract Full Text PDF PubMed Google Scholar). were at and with the cells were by at for with of and at for The cells were in 1 and by a at The was at for The was of with The was in a with of and λPP with containing containing λPP were and by the of to at the protein was in and to a with The was with of the and with of The was with of in were using as a substrate, and at in and The S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) was cells and used to of for at were used to 5 of 2 in a were at at of to a that an at of was to a of and the cells were with 1 Another of was to a of the to an at of The cells were by at for The was in of of cells, this and were with on for by a to the the and were with on at for 1 the protein was as for wild type were at on a A of was used for were from the is the is the in is the and is the of in is in of calcineurin and were in 1 phosphatase activity of calcineurin and was using as a substrate at in MOPS, 1 and wild type calcineurin type λPP and were at in DTT, 1 and wild type λPP for 5 at were by the of activity was by the in at with using at and at based on a of and for the The of was from 2 to and the kinetic kcat were by the to the using a was phosphorylated with to a activity of using the catalytic subunit of protein and as M.J. J. A Scholar). were as M.J. J. A Scholar) in MOPS, 1 DTT, and peptide was with peptide and substrate in from to 1 Calcineurin were for wild type and for were in and to the by a were by the of phosphate of phenyl phosphate as PubMed Scopus Google Scholar). were in containing 1 and phosphate were from 1 to for λPP and 1 to for type protein from to from to was 5 and by the of phenyl from to of the was and to of a containing a of to in 1 of was and the at phosphate was from a using of Kinetic for λPP and were as in DTT, in was to and by with was to a of and to a of The were with and an at were using in an to 2 with MOPS, 1 and using to were to EPR and in of wild type calcineurin and λPP were in a similar that at for and both enzymes were 1 to the the iron-reconstituted EPR of CN(H151Q), a of was to a of for 5 at and in EPR were using a at with an for were from was by the using The of in calcineurin to the dinuclear metal center can be in was mutated to a glutamine by mutagenesis to the on activity and of the dinuclear metal The mutant calcineurin A subunit was in E. in a to that of the wild type calcineurin A subunit and with calcineurin to the mutant protein The of the as as the of in the calcineurin A gene were by sequence the of was less that for the wild type be and to for and spectroscopic A 1 of of The analogous in λPP, identified by sequence comparisons as (7Lohse D.L. Denu J.M. Dixon J.E. Structure. 1995; 3: 987-990Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), was mutated to an asparagine S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). In this the protein was to as to of of wild type calcineurin and for a of from to of both wild type calcineurin and to of calcineurin D.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) not Kinetic using and as for wild type calcineurin and in the of 1 compared in I. Using either substrate, the for the mutant were the kcat for wild type the kcat for using as a substrate, is wild type calcineurin in an Using [P]-RII peptide, for wild type calcineurin and were and respectively, a of Using [P]-RII for both of calcineurin were the within the of the a decrease in was for compared with wild type calcineurin using as a for wild type and calcineurin using and [P]-RII peptide as kcat compared with wild type calcineurin for substrate type 1 type 1 1 were as for kinetic were by a with the in the were from at kcat compared with wild type calcineurin for substrate in a Kinetic were as for kinetic were by a with the in the were from at Kinetic using and phenyl phosphate as for wild type λPP and compared in These were also by of in Using as substrate, the kcat for wild type λPP and were and a 590-fold is to the decrease in kcat for calcineurin for the analogous The inkcat for λPP is less the S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) of a activity for the protein in the Using phenyl phosphate as substrate, for λPP was compared with for λPP(H76N), a of The were similar for both mutant and wild type proteins using either for λPP and histidine mutant using and phenyl phosphate as kcat compared with wild type λPP for substrate type 1 S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google phosphate type of λPP and were as with and phenyl phosphate as of wild type and mutant λPP using as a substrate for the the from a with the in the were from at kcat compared with wild type λPP for substrate in a Kinetic of λPP and were as with and phenyl phosphate as of wild type and mutant λPP using as a substrate for the the from a with the in the were from at not a metal in calcineurin is to either metal that mutagenesis the the metal the of mutagenesis on the dinuclear center, wild type calcineurin and were with to a as a spectroscopic of the active site and analyzed by EPR The EPR of wild type CN(H151Q), and in the of phosphate compared in in the EPR a of and not for a and a the of the EPR signal in 2 A to an of and signal is to the signal in calcineurin with and from a dinuclear center in the L. Haddy F. J. Chem. 1995; Scopus (59) Google Scholar). The that the of the dinuclear center 1 is the is the is the and the for metal between the and of the a with to this EPR 2 the EPR of iron-reconstituted EPR signal of a dinuclear center in the is and that a dinuclear metal center can be in the mutant is the signal from the center in wild type of the EPR of and of with the of a dinuclear center but also the of a metal center either of to the EPR in the that the signal in 2 from an active site metal center was by phosphate to the The of phosphate to a of the EPR signal 2 of the was that phosphate to both of the metal of the dinuclear center. In a similar to that for λPP and were with to a spectroscopic of the active site metal λPP as as by metal using of of and of of also of of of and of of of wild type λPP and with that EPR with 3, A and of both EPR of protein and of protein for wild type λPP, and of protein and of protein for both λPP and can accommodate dinuclear metal in the of the EPR of the mutant is from the of wild type of calcineurin (11Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (762) Google Scholar, F. J.E. J.E. Nature. 1995; PubMed Scopus Google Scholar), PP1 (9Goldberg J. Huang H.-b. Kwon Y.-g. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (735) Google M.-P. Cohen P.T.W. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (373) Google Scholar), and phosphatase B. J. Mol. Biol. 1996; PubMed Scopus Google Scholar, P. B. 1995; 268: PubMed Scopus Google Scholar) have identified a conserved histidine in active site although not to either metal of the dinuclear metal center, is within 5 Å of both metal In this study we have mutated the of calcineurin to glutamine to in and on the active site dinuclear metal center. analogous histidine in the bacteriophage λ protein phosphatase has been to asparagine S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The resulted in a inkcat pNPP, a in for the metal used in for substrate. In a study by and J. K. 1996; PubMed Scopus Google Scholar), the in PP1 was mutated to a number of In that study was that of the resulted in the of protein for and a of the protein was and be by PP1 mutant phosphatase the for activity to wild type PP1 was not to the in the PP1 the of of protein was also that for wild type calcineurin the of the was wild type in an L. and F. with wild type calcineurin F. Res. 1994; PubMed Scopus Google Scholar), of E. the calcineurin A subunit at the of protein in of the mutant calcineurin A subunit with calcineurin of protein for studies and spectroscopic using the of mutagenesis on catalytic were used for and is a substrate for calcineurin J. Biol. Chem. Full Text PDF PubMed Google Scholar), the of is the of for [P]-RII calcineurin activity using [P]-RII peptide is by the immunosuppressant drug cyclosporin and is using (1Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3546) Google Scholar, F. Sci. S. 1992; PubMed Scopus Google Scholar). calcineurin phosphatase activity [P]-RII peptide is in the of of with an to the for not These that both and [P]-RII peptide the active and a of kinetic is was in to the activity for both enzymes (5Zhuo S. Clemens J.C. Hakes D.J. Barford D. Dixon J.E. J. Biol. Chem. 1993; 268: 17754-17761Abstract Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is known to both metal in PP1 (9Goldberg J. Huang H.-b. Kwon Y.-g. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (735) Google Scholar, 10Egloff M.-P. Cohen P.T.W. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (373) Google Scholar). that a dinuclear is also in Yu and F. In of catalytic mutagenesis of to glutamine resulted in a decrease in kcat using as substrate. In the kcat for 1,300-fold compared with wild type calcineurin using [P]-RII peptide as substrate. either was either not by The kcat for wild type calcineurin in this study F. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, B. D.J. J. Biol. Chem. Full Text PDF PubMed Google differences between Using as substrate, also in substrate and a decrease inkcat of 590-fold to wild type is the S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) of a activity for the mutant in this study but also of a activity of the wild type used in this study In inkcat has been for wild type λPP (5Zhuo S. Clemens J.C. Hakes D.J. Barford D. Dixon J.E. J. Biol. Chem. 1993; 268: 17754-17761Abstract Full Text PDF PubMed Google S. Clemens J.C. Dixon J.E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, S. Sci. S. 1993; PubMed Scopus (42) Google we have also a in activity for of wild type λPP by similar Kinetic of wild type λPP and for a substrate, phenyl the using a decrease inkcat of 530-fold and a to wild type on sequence λPP is to be a member of the family of PP1, and (6Koonin E.V. Prot. Sci. 1994; 3: 356-358Crossref PubMed Scopus (105) Google Scholar). that λPP can accommodate a metal center as for enzymes containing the is that in λPP and in calcineurin have similar in phosphate for this conserved histidine include an active site a in substrate, a in of the a in by of an solvent molecule. of be with the decrease in activity for wild type mutant enzymes. The for to the decrease of for and for the of phosphatase J.E. J.A. J. Chem. 1992; Scopus Google Scholar). with a member of the with a similar active site as calcineurin F. Yu L. Mertz P. J. Biol. Inorg. Chem. 1996; 1: 388-396Crossref Scopus (42) Google Scholar) that by of the substrate to solvent J. Chem. 1993; Scopus Google Scholar). that this histidine in substrate for mutant and wild type enzymes also a in substrate In the of calcineurin with phosphate at the active is within of the of to in (11Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (762) Google Scholar). in the of with M.-P. Cohen P.T.W. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (373) Google Scholar), with phosphate in the active site (9Goldberg J. Huang H.-b. Kwon Y.-g. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (735) Google Scholar), and in the phosphatase with phosphate and at the active site B. J. Mol. Biol. 1996; PubMed Scopus Google Scholar). were in to the for mutant to wild type a using that have of In a in kcat for peptide is the of the have that by has a of compared with for With λPP, is between kcat of wild using and phenyl phosphate It that this histidine is not for of the the kcat for and compared with wild type the of for of pNPP, at J. Chem. Scopus Google Scholar). of phosphate with a of at The Scholar). A of this catalytic activity is derived from the of the active site dinuclear metal center, has been to the of a the in the be in with this solvent to either position a on the for on the of the substrate to as a to a with solvent in of the of was to of solvent to the F. J.E. J.E. Nature. 1995; PubMed Scopus Google Scholar). In the of PP1 with the of the analogous was also within of a but that was Å from the metal (9Goldberg J. Huang H.-b. Kwon Y.-g. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (735) Google Scholar). for this is by mutagenesis studies of PP1 the of a conserved on catalytic conserved is of the consensus motif (6Koonin E.V. Prot. Sci. 1994; 3: 356-358Crossref PubMed Scopus (105) Google Scholar, 7Lohse D.L. Denu J.M. Dixon J.E. Structure. 1995; 3: 987-990Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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The EPR of iron-reconstituted with the of a that the mutant is to a dinuclear metal center. the of this was from that of wild type calcineurin. In the EPR of the iron-reconstituted also the signal with a from the of wild type The that phosphate to iron-reconstituted a in the of the EPR that from an active site metal center. in both wild type calcineurin and phosphate binding to the to a of the EPR a of a decrease in the by the phosphate the metal of the J. Jr., L. J. Biol. Chem. Full Text PDF PubMed Google With CN(H151Q), on the phosphate a of the EPR spectroscopic is to the for These demonstrate that the conserved histidine in the calcineurin and λPP is an essential of the active site disruption to in of activity have resulted from of an active site and the disruption of an essential bond to a metal-coordinated solvent molecule. to this in for the containing the for λPP wild type and also and at for the of Kuriyan and for PP1 and and for with Kinetic and spectroscopic of of a conserved histidine in the calcineurin and λ protein of The in and the and the decrease in activity of mutant compared to wild PDF