Study of the action of human salivary alpha-amylase on 2-chloro-4-nitrophenyl α-maltotrioside in the presence of potassium thiocyanate

Article abstract of DOI:10.1016/S0008-6215(97)00150-X

The degradation mechanism of a synthetic substrate, 2-chloro-4-nitrophenyl α-maltotrioside (CNP-G₃), by human salivary alpha-amylase (HSA) was investigated by kinetic and product analyses. It was observed that the enzyme attacked the various CNP-maltooligosaccharides (CNP-G₃, to CNP-G₆) releasing free CNP. Addition of 500 mM potassium thiocyanate (KSCN) was also found to greatly increase the rates of CNP-release. It was the fastest with CNP-G₃, and, in the presence of KSCN, was almost comparable to that of degradation of maltopentaose (G₅). On the other hand, addition of KSCN decreased the rate of cleavage between glucan-glucan bonds in maltopentaose. Product analysis showed that KSCN addition altered the cleavage distribution which occurred 100% at the bond between CNP and G₃, and that product distribution of free CNP was largely dependent on substrate concentration. Formation of CNP-G₆, a larger product than the original substrate CNP-G₃, was found to be present in the digest at high concentrations of substrate and in the presence of KSCN. Based on these results, a degradation pathway for CNP-G₃ involving transglycosylation besides direct hydrolysis is proposed. The increase of the CNP-release by the addition of KSCN would result from a corresponding increase in the interaction between the CNP moiety and the corresponding subsite near the catalytic site, as well as the enhancement of the catalytic efficiency.