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 A 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 A protein phosphatase accommodates a dinuclear metal center. Calcineurin and A 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 A 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 A 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 A protein phosphatase mutant, k(cat) decreased 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.