Crystallographic evidence for substrate-assisted catalysis in a bacterial beta-hexosaminidase

beta -Hexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of beta -1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates, Heritable deficiency of this enzyme results in various fo rms of GalNAc-beta (1,4)-[N-acetylneuraminic acid (2,3)]-Gal-beta (1,4)-Glc -ceramide gangliosidosis, including Tay-Sachs disease. We have determined t he x-ray crystal structure of a P-hexosaminidase from Streptomyces plicatus to 2.2 Angstrom resolution (Protein Data Bank code 1HP4), P-Hexosaminidase s are believed to use a substrate-assisted catalytic mechanism that generat es a cyclic oxazolinium ion intermediate. We have solved and refined a comp lex between the cyclic intermediate analogue N-acethlglucosamine-thiazoline and beta -hexosaminidase from S, plicatus to 2.1 Angstrom resolution (Prot ein Data Bank code 1HP5), Difference Fourier analysis revealed the pyranose ring of N-acetylglucosamine-thiazoline bound in the enzyme active site wit h a conformation close to that of a C-4(1) chair. A tryptophan-lined hydrop hobic pocket envelops the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within this pocket, Tyr(393) and Asp(313) appear i mportant for positioning the 2-acetamido group of the substrate for nucleop hilic attack at the anomeric center and for dispersing the positive charge distributed into the oxazolinium ring upon cyclization, This complex provid es decisive structural evidence for substrate-assisted catalysis and the fo rmation of a covalent, cyclic intermediate in family 20 beta -hexosaminidas es.