New insight into enzymatic hydrolysis of peptides with site-specific amino acid D-isomerization

Abstract The isomerization of L-amino acids in peptides and proteins into D-configuration under physiological conditions would affect the physiological dysfunction and caused protein conformational diseases. The presence of D-amino acids might change the higher-order structure of proteins and triggered abnormal aggregation. In order to better understand this phenomenon and promote degradation, we systematically studied the enzymatic hydrolysis of a series of peptides obtained by replacing L-amino acids in different positions of template peptide KYNETWRSED with D-amino acids under the action of Protease K. The results showed that, compared with normal peptide, isomerization of different amino acids had different effects on the anti-enzymatic hydrolysis of the peptides, especially D-tryptophan at position 6, which significantly inhibited enzymatic hydrolysis. The analysis of the peptide cleavage site revealed that the efficiency of enzymatic hydrolysis mainly depended on the isomerization of the amino acids at a specific site of the peptide cleavage. Further studies showed that the enzymatic hydrolysis of substrates could be facilitated by optimized reaction conditions such as temperature, pH, addition of metal ions, and change of buffer. In this way the accumulation of disease-associated D-amino acid containing polypeptides/proteins could be prevented.