STUDY OF THE ACTION OF HUMAN SALIVARY ALPHA-AMYLASE ON 2-CHLORO-4-NITROPHENYL ALPHA-MALTOTRIOSIDE IN THE PRESENCE OF POTASSIUM THIOCYANATE

The degradation mechanism of a synthetic substrate, 2-chloro-4-nitroph enyl alpha-maltotrioside (CNP-G(3)), 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-G (3) to CNP-G(6)) releasing free CNP. Addition of 500 mM potassium thio cyanate (KSCN) was also found to greatly increase the rates of CNP-rel ease. It was the fastest with CNP-G(3), and, in the presence of KSCN, was almost comparable to that of degradation of maltopentaose (G(5)). On the other hand, addition of KSCN decreased the rate of cleavage bet ween glucan-glucan bonds in maltopentaose. Product analysis showed tha t KSCN addition altered the cleavage distribution which occurred 100% at the bond between CNP and G(3), and that product distribution of fre e CNP was largely dependent on substrate concentration. Formation of C NP-G,, a larger product than the original substrate CNP-G,, 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 f or CNP-G(3) involving transglycosylation besides direct hydrolysis is proposed. The increase of the CNP-release by the addition of KSCN woul d result from a corresponding increase in the interaction between the CNP moiety and the corresponding subsite near the catalytic site, as w ell as the enhancement of the catalytic efficiency. (C) 1997 Elsevier Science Ltd.