title: Implication of tryptophan and histidine in the active site of endo-polygalacturonase from Aspergillus ustus: elucidation of the reaction mechanism.
abstract: The mechanism of action for the hydrolysis of polygalacturonic acid by the enzyme endo-polygalacturonase (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) was investigated. The enzyme from Aspergillus ustus was purified to homogeneity and used for the study. The endo-polygalacturonase had a molecular weight of 36,000 daltons, a pI of 8.3, specific activity of 785 units/mg, Km of 0.82 mg/ml, and Vmax of 976 micromoles of product min-1 mg-1. Amino acids involved in the catalysis were identified by chemical modification and the active site characterized. Inhibition by hydroxynitrobenzyl bromide and diethylpyrocarbonate, followed by substrate protection studies showed that tryptophan and histidine were involved at or near the active site. Kinetic constants of partially inhibited enzyme, suggest the involvement of tryptophan in substrate binding and histidine in catalysis. Quenching of tryptophan fluorescence of the enzyme in the presence of polygalacturonic acid substantiated the conclusion that tryptophan was involved in substrate binding. An isotope effect of 1.8 was observed with deuterated water on the Vmax of the endo-polygalacturonase, with the proton inventory giving a linear relationship. The proposed mechanism involves a single proton transfer from the histidine residue of the enzyme to the glycosidic oxygen and hydrolysis by the addition of a water molecule.
authors.affiliation: Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
authors.forename: M N
authors.initials: MN
authors.lastname: Rao
authors.forename: A A
authors.initials: AA
authors.lastname: Kembhavi
authors.forename: A
authors.initials: A
authors.lastname: Pant
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D001665:Binding Sites; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry / Q000502:physiology*; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D011096:Polygalacturonase / Q000737:chemistry* / Q000302:isolation & purification; D014364:Tryptophan / Q000737:chemistry / Q000502:physiology*
chemical_list: D005656:Fungal Proteins; D006639:Histidine; D014364:Tryptophan; D011096:Polygalacturonase