Mechanism of hydrolyses of phenyl alpha-maltosides catalyzed by taka-amylase A.
Michaelis constants (Kms) and molecular activities (kos) of phenyl, p-nitrophenyl and p-methylphenyl alpha-maltoside for taka-amylase A catalyzed hydrolyses were determined in H2O and in D2O at pH or pD 5.3 and at 25 degrees C. Production of alpha-maltose in the hydrolysis was confirmed by 1H NMR. Neither substituent nor solvent deuterium isotope effects on Kms for phenyl, p-nitrophenyl and p-methylphenyl alpha-maltosides were detected. On the other hand, substituent effects on kos of these compounds were evident, but the isotope effects on kos were not marked, so that protonation of the substrate in the catalytic reaction might not be rate-limiting. The result indicates that nucleophilic attack of a carboxylate anion of the enzyme upon the protonated substrate is the rate-limiting step in the hydrolysis proceeding through the nucleophilic double displacement mechanism, which involves a covalently bonded glycosyl intermediate. The molecular orbitals of phenyl alpha-D-glucosides as model compounds of phenyl alpha-maltosides were calculated by the AM1 method. From the results, it was concluded that the lowering of the lowest unoccupied molecular orbital (LUMO) energy levels and the increase of distribution of LUMO on the anomeric carbon, C-1, of the compounds are caused by protonation at the glycosidic oxygen from the protonated carboxyl group of the enzyme. This causes acceleration of the hydrolysis of a substrate by the enzyme.
Isoda
Y
Y
Department of General Education, Osaka Institute of Technology.
Shimizu
Y
Y
Hashimoto
A
A
Fujiwara
H
H
Nitta
Y
Y
Kagemoto
A
A
D006027:Glycosides / Q000737:chemistry*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D000516:alpha-Amylases / Q000737:chemistry*
D006027:Glycosides; D000516:alpha-Amylases