Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products.
Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolase-derived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the K(M) value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on V(max) and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.
Pietkiewicz
Jadwiga
J
Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland.
Gamian
Andrzej
A
Staniszewska
Magdalena
M
Danielewicz
Regina
R
D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011765:Pyruvaldehyde / Q000494:pharmacology*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D017127:Glycation End Products, Advanced; D011765:Pyruvaldehyde; D010751:Phosphopyruvate Hydratase