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Chloride promotes refolding of active Vibrio alkaline phosphatase through an inactive dimeric intermediate with an altered interface.
FEBS Open Bio 2019 January
Most enzymes are homodimers or higher order multimers. Cold-active alkaline phosphatase from Vibrio splendidus (VAP) transitions into a dimer with very low activity under mild denaturation conditions. The desire to understand why this dimer fails to efficiently catalyse phosphomonoester hydrolysis led us to investigate interfacial communication between subunits. Here, we studied in detail the unfolding mechanism at two pH values and in the presence or absence of sodium chloride. At pH 8.0, the denaturation model had to include an inactive dimer intermediate and follow the pathway: N 2 → I 2 → 2 U . At pH 10.5, the model was of a two-state nature. Enzyme activity was not recovered under several examined refolding conditions. However, in the presence of 0.5 m NaCl, the enzyme was nearly fully reactivated after urea treatment. Thermal inactivation experiments were biphasic where the inactivation could be detected using CD spectroscopy at 190-200 nm. By incorporating a bimane fluorescence probe at the dimer interface, we could monitor inactivation/denaturation at two distinct sites at the dimer interface. A change in bimane fluorescence at both sites was observed during inactivation, but prior to the global unfolding event. Furthermore, the rate of change in bimane fluorescence correlated with inactivation rates at 40 °C. These results indicate and support the hypothesis that the subunits of VAP are only functional in the dimeric state due to the cooperative nature of the reaction mechanism when proper crosstalk between subunits is facilitated.
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