title: Kinetic analysis and mechanism on the inhibition of chlorogenic acid and its components against porcine pancreas alpha-amylase isozymes I and II.
abstract: Chlorogenic acid (5-caffeoylquinic acid, 5-CQA) is a kind of polyphenol and is richly included in green coffee beans. The inhibitory effects of 5-CQA and its components, caffeic acid (CA) and quinic acid (QA), on the two porcine pancreas alpha-amylase (PPA) isozymes, PPA-I and PPA-II, were investigated using p-nitrophenyl-alpha-D-maltoside as substrate at pH 6.9 and 30 degrees C. The inhibition potencies of the respective inhibitors against both PPA isozymes were almost the same and in the order of 5-CQA > CA >> QA. Their IC(50) values were 0.07-0.08 mM, 0.37-0.40 mM, and 25.3-26.5 mM, respectively. The inhibition mechanisms of 5-CQA and CA were investigated by kinetic analyses, and the inhibitor constants K(i) and K(i)' (for the free enzyme and enzyme-substrate complex, respectively) were determined. It was indicated that 5-CQA and CA showed mixed-type inhibition with K(i) > K(i)' against both PPA-I and PPA-II. The binding of PPA-I or PPA-II with 5-CQA or CA was all exothermic and enthalpy-driven. QA is a poor inhibitor, and its inhibitory mode was unique and hardly analyzed by a simple Michaelis-Menten-type interaction between the enzyme and inhibitor. However, it was shown that the inhibitory activity of CA was enhanced 5 times by ester-bond formation with QA in the form of 5-CQA. These results provide us with significant hints for the development of alpha-amylase inhibitors useful for the prevention of diabetes and obesity.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Narita
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D000818:Animals; D002726:Chlorogenic Acid / Q000378:metabolism / Q000494:pharmacology*; D040503:Coffea / Q000737:chemistry; D003924:Diabetes Mellitus, Type 2 / Q000517:prevention & control; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D009765:Obesity / Q000517:prevention & control; D010179:Pancreas / Q000201:enzymology*; D012639:Seeds / Q000737:chemistry; D013552:Swine; D013816:Thermodynamics; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D002726:Chlorogenic Acid; D000516:alpha-Amylases