Hydrolytic enzyme substrates. 3. Phosphomonoesterase substrates

This report documents the use of a new and sensitive colorimetric method for measuring phosphomonoesterase activity. The substrates are the phosphate esters of 4-(p-nitrophenoxy)-1,2-butanediol (PNB), 4-(2,4-dinitrophenoxy)-1,2-butanediol (DNB) and 3-(p-nitrophenoxy)-1,2-propanediol (PNG). The key intermediate in the assay is the nitrophenoxy diol which is obtained by enzyme hydrolysis of its phosphate ester. Periodate oxidation of this substance in solution containing methylamine quantitatively yields its nitrophenolate ion whose concentration is determined colorimetrically. The amount of nitrophenolate ion is thus equivalent to the amount of nitrophenoxy diol whose concentration is a function of the phosphomonoesterase activity in the assay sample. The unhydrolyzed phosphomonoester is completely stable to periodate and the hydrolytic conditions used in the assay. The enzymes used to test the substrates were E. coli alkaline phosphomonoesterase and wheat germ phosphomonoesterase. These new esters were all better substrates than the glycerol phosphate esters. Their Michaelis-Menten constants were determined for E. coli phosphomonoesterase.