Protein Amadoriase Activity of the Escherichia coli K-12 Glycolytic Enzyme Phosphoglucose Isomerase

Abstract

The Maillard reaction (glycation) is a spontaneous non-enzymatic reaction between primary amines and carbonyl compounds, which affects proteins and DNA of both pro- and eukaryotes. In recent studies, we have shown that the glycolytic enzyme of Escherichia coli phosphoglucose isomerase (PGI) catalyzes in vitro the deglycation of DNA modified with glucose 6-phosphate (G6P)-derived Amadori products (APs). APs are early products of the Maillard reaction, which are formed not only on DNA but also on other amines including proteins. The aim of the current study was to test the E. coli PGI for protein deglycation (amadoriase) activity. To this end, we used chicken lysozyme glycated with G6P as a model protein. Treatment of the glycated lysozyme with protein extract from an E. coli PGI proficient but not deficient strain resulted in the release of G6P, which was indicative of PGI protein amadoriase activity. G6P-derived APs represent fructose 6-phosphate (F6P) residues bound to free amino groups of the model protein and because of that we compared the kinetic constants of the E. coli PGI for the glycated lysozyme and for free F6P. PGI demonstrated nearly two times higher affinity to the glycated lysozyme (K_m' = 0.06 mM) than to free F6P (K_m' = 0.1 mM). However, the apparent catalytic constant of the enzyme with the glycated lysozyme (K_cat' = 93 s^–1) was eight times lower than with F6P (K_cat' = 736 s^–1). Future studies are expected to shed light on the physiological relevance of the PGI protein amadoriase activity we report here.