Reactive carbonyls such as 4-hydroxy-2-nonenal (4-HNE), trans-2-nonenal (T2 N), acrolein (ACR) can react readily with nucleophilic protein sites forming of advanced glycation end-products (AGE). In this study, the human and pig muscle-specific enolase was used as a protein model for in vitro modification by 4-HNE, T2 N and ACR. While the human enolase interaction with reactive α-oxoaldehyde methylglyoxal (MOG) was demonstrated previously, the effect of 4-HNE, T2N and ACR has not been identified yet. Altering in catalytic function were observed after the enzyme incubation with these active compounds for 1-24 h at 25, 37 and 45 °C. The inhibition degree of enolase activity occurred in following order: 4-HNE > ACR > MOG > T2N and inactivation of pig muscle-specific enolase was more effective relatively to human enzyme. The efficiency of AGE formation depends on time and incubation temperature with glycating agent. More amounts of insoluble AGE were formed at 45 °C. We found that pyridoxamine and natural dipeptide carnosine counteracted AGE formation and protected enolase against the total loss of catalytic activity. Moreover, we demonstrated for the first time that phosphatidylserine may significantly protect enolase against decrease of catalytic activity in spite of AGE production.