pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium.

Abstract:

:The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7-10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the Ki for inorganic phosphate (product inhibition) and the KM for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and urea-induced inactivation was shown to be accompanied by neither dissociation of the active site metal ions nor dimer dissociation. This would suggest that the inactivation involved subtle changes in active site conformation. Furthermore, the VAP dimer equilibrium was studied for the first time and shown to highly favor dimerization, which was dependent on pH and NaCl concentration. Taken together, the data support a model in which anions bind to some specific acceptor in the active site of VAP, resulting in great stabilization and catalytic rate enhancement, presumably through a different mechanism.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Hjörleifsson JG,Ásgeirsson B

doi

10.1021/acs.biochem.7b00690

subject

Has Abstract

pub_date

2017-09-26 00:00:00

pages

5075-5089

issue

38

eissn

0006-2960

issn

1520-4995

journal_volume

56

pub_type

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