Interaction of Bacteriophage λ Protein Phosphatase with Mn(II): Evidence for the Formation of a [Mn(II)]₂ Cluster^†

The interaction of bacteriophage λ protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry. Reconstitution of bacteriophage λ protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein. This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2. The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster. EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependent studies indicated that these features arose from an excited state. Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity. The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites. These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases

The Transition State of the Phosphoryl-Transfer Reaction Catalyzed by the Lambda Ser/Thr Protein Phosphatase

The catalytic reaction of the Mn2+ form of the native bacteriophage λ phosphatase and the H76N mutant was studied with the substrate p-nitrophenyl phosphate using heavy atom isotope effects and pH-dependent rate studies. The kinetic isotope effects in the substrate were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom undergoing bond cleavage [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects with native enzyme at the pH optimum of 7.8 were 1.0133 ± 0.0006 for 18(V/K)bridge, 1.0006 ± 0.0003 for 15(V/K), and 0.9976 ± 0.0003 for 18(V/K)nonbridge. These values were constant within experimental error across the pH range from 6.0 to 9.0 and were also unchanged for the slower catalytic reaction resulting when Ca2+ was substituted for Mn2+. The results indicate that the chemical step of P−O bond cleavage is rate-limiting, the first metallophosphatase for which this has been shown to be the case. The isotope effects are very similar to those measured for reactions of protein-tyrosine phosphatases, indicating that the two families of enzymes share similar dissociative transition states. The 18(V/K)bridge and 15(V/K) isotope effects for the H76N mutant were slightly increased in magnitude relative to the native enzyme but were much smaller than the values expected if the leaving group were departing with a full negative charge. The pH vs kcat profile for the native enzyme is bell-shaped with pKa values of 7.7 ± 0.3 and 8.6 ± 0.4. Km values for substrate increased with pH approximately 70-fold across the pH range 5.8−9.1. The Km for the H76N mutant was similar to that observed for native enzyme at high pH and was relatively constant across this pH range. The basic limb of the pH−rate profile is reduced but not abolished in the H76N mutant reaction. The results are discussed in terms of the possible role of His-76 and the nature of the transition state for catalysis in the native enzyme and mutant

Supplemental Material, sj-pdf-5-jpx-10.1177_23743735211034032 - Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs

Supplemental Material, sj-pdf-5-jpx-10.1177_23743735211034032 for Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs by Karen Homa, Gabrielle Stevens, Rachel Forcino, Peter Scalia, Pamela Mertz and Glyn Elwyn in Journal of Patient Experience</p

Supplemental Material, sj-pdf-2-jpx-10.1177_23743735211034032 - Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs

Supplemental Material, sj-pdf-2-jpx-10.1177_23743735211034032 for Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs by Karen Homa, Gabrielle Stevens, Rachel Forcino, Peter Scalia, Pamela Mertz and Glyn Elwyn in Journal of Patient Experience</p

Supplemental Material, sj-pdf-3-jpx-10.1177_23743735211034032 - Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs

Supplemental Material, sj-pdf-3-jpx-10.1177_23743735211034032 for Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs by Karen Homa, Gabrielle Stevens, Rachel Forcino, Peter Scalia, Pamela Mertz and Glyn Elwyn in Journal of Patient Experience</p

Supplemental Material, sj-pdf-1-jpx-10.1177_23743735211034032 - Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs

Supplemental Material, sj-pdf-1-jpx-10.1177_23743735211034032 for Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs by Karen Homa, Gabrielle Stevens, Rachel Forcino, Peter Scalia, Pamela Mertz and Glyn Elwyn in Journal of Patient Experience</p

Supplemental Material, sj-pdf-4-jpx-10.1177_23743735211034032 - Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs

Supplemental Material, sj-pdf-4-jpx-10.1177_23743735211034032 for Assessing Shared Decision-Making in Cystic Fibrosis Care Using collaboRATE: A Cross-Sectional Study of 159 Programs by Karen Homa, Gabrielle Stevens, Rachel Forcino, Peter Scalia, Pamela Mertz and Glyn Elwyn in Journal of Patient Experience</p