Detailed kinetic analysis and identification of the nucleophile in alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase.
alpha-l-Arabinofuranosidases cleave the l-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-alpha-l-arabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl alpha-l-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K(m) value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2',4',6'-trichlorophenyl alpha-l-arabinofuranoside by the E294A mutant. The glycosyl-azide product formed during this reaction was isolated and characterized as beta-l-arabinofuranosyl-azide by (1)H NMR, (13)C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the alpha-l-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.
Shallom
Dalia
D
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Belakhov
Valery
V
Solomon
Dmitry
D
Shoham
Gil
G
Baasov
Timor
T
Shoham
Yuval
Y
D000409:Alanine / Q000737:chemistry; D001386:Azides / Q000494:pharmacology; D001406:Bacillaceae / Q000201:enzymology*; D020134:Catalytic Domain; D002473:Cell Wall / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D018698:Glutamic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D019810:Sodium Azide / Q000494:pharmacology; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors
D001386:Azides; D018698:Glutamic Acid; D019810:Sodium Azide; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase; D000409:Alanine