Determination of endopolyphosphatase using polyphosphate glucokinase

Area of Science:

• Biochemistry
• Enzymology

Background:

• Endopolyphosphatase (EC 3.6.1.10) activity is challenging to quantify due to chemically identical substrates and products.
• Existing viscometric methods lack sensitivity and are unsuitable for short polyphosphate substrates.

Purpose of the Study:

• Develop a sensitive and rapid method for determining endopolyphosphatase activity.
• Differentiate endopolyphosphatase from exopolyphosphatase (EC 3.6.1.11) activity.

Main Methods:

• Utilized polyphosphate glucokinase (EC 2.7.1.63) to convert polyphosphates of varying sizes into a uniform short-chain product (poly(P)m).
• Monitored the increase in poly(P)m concentration, measured as acid-labile phosphorus, which is proportional to endopolyphosphatase activity.

Main Results:

• The assay provides a relative measure of endopolyphosphatase activity based on the increase in poly(P)m concentration.
• Under specific conditions, poly(P)m can be quantified as poly(P)3.5, allowing for calculation of the exact number of polyphosphate chains formed.
• The method is rapid, simple, sensitive, and distinguishes between endo- and exopolyphosphatase activities.

Conclusions:

• The developed method accurately quantifies endopolyphosphatase activity.
• This assay offers significant advantages over existing methods in terms of sensitivity, substrate versatility, and specificity.