Abstract

Saccharomyces cerevisiae calcineurin (CN) consists of a catalytic subunit CNA1 or CNA2 and a regulatory subunit CNB1. The kinetics of activation of yeast CN holoenzymes and their catalytic domains by Mn²⁺ were investigated. We report that the in vitro phosphatase reaction activated by Mn²⁺ typically has a pronounced initial lag phase caused by slow conformational rearrangement of the holoenzyme-Mn²⁺. A similar lag phase was detected using just the catalytic domain of yeast CN, indicating that the slowness of Mn²⁺-induced conformational change of CN results from a rearrangement within the catalytic domain. The Mn²⁺-activation of CN was reversible. The dissociation constant of the CN heterodimer containing the CNA2 subunit in the presence of Mn²⁺ was 3-fold higher than that of CN containing the CNA1 subunit and that of the catalytic domains of CNA1 and CNA2, pointing to differences between the residues surrounding the Mn²⁺-binding sites of CNA1 and CNA2.