Inhibitory effect of glycation on catalytic activity of alanine aminotransferase

Non-enzymatic glycation is a common post-translational modification of tiss ue and plasma proteins which can impair their functions in living organisms . In this study, the authors have demonstrated for the first time an inhibi tory effect of in vitro glycation on the catalytic activity of alanine amin otransferase (ALT, EC 2.6.1.2), a pyridoxal phosphate enzyme with several l ysine residues in the molecule. The porcine heart enzyme was incubated with 50 mmol/l D-fructose, D-glucose, D,L-glyceraldehyde, or D-ribose in 0.1 mo l/l phosphate buffer (pH 7.4) at 25 degreesC for up to 20 days. The stronge st glycation effect was shown by D,L-glyceraldehyde, which caused complete enzyme inhibition within 6 days. After 20 days of incubation, the ALT activ ity in samples with D-fructose and D-ribose was less than 7% of the initial enzyme activity. A statistically significant effect of D-glucose on the en zymatic activity of ALT was not found. Incubation of ALT with D-fructose, D ,L-glyceraldehyde and D-ribose minimized its catalytic activity both in the glycated and non-glycated fractions of the samples. Markedly higher activi ty was found in the glycated fraction with glucose. The inhibitory effect o f glycation of ALT with D-fructose and D-ribose was found to be more intens ive in the presence of L-alanine and weaker in the presence of 2-oxoglutara te. The findings suggest that glycation of the e-amino group of Lys313 as a crucial part of the catalytic site of ALT may contribute to ALT inactivati on in the presence of glycating sugars. Nevertheless, glycation of lysine r esidues outside the active center of ALT seems to be primary.