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-nitrophenyl alpha-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.
Suganuma
T
T
Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Japan.
Maeda
Y
Y
Kitahara
K
K
Nagahama
T
T
D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D012463:Saliva / Q000201:enzymology*; D013861:Thiocyanates / Q000378:metabolism*; D014312:Trisaccharides / Q000378:metabolism*; D000516:alpha-Amylases / Q000378:metabolism*
D004396:Coloring Agents; D009844:Oligosaccharides; D013861:Thiocyanates; D014312:Trisaccharides; C057413:2-chloro-4-nitrophenylmaltotrioside; C019193:maltopentaose; D000516:alpha-Amylases; C009941:potassium thiocyanate