Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Alkaline Phosphatase.

Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species.

The high catalytic rate of the cold-active Vibrio alkaline phosphatase requires a hydrogen bonding network involving a large interface loop.

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability.

X-ray crystal structure of alkaline phosphatase with the non-competitive inhibitor cyclohexylamine.

Background: -nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs.

Methods: Cyclohexylamine inhibited four different APs.

Chloride promotes refolding of active alkaline phosphatase through an inactive dimeric intermediate with an altered interface.

Most enzymes are homodimers or higher order multimers. Cold-active alkaline phosphatase from (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.

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

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).