IDENTIFICATION OF 2 ASPARTATES AND A GLUTAMATE ESSENTIAL FOR THE ACTIVITY OF ENDO-BETA-N-ACETYLGLUCOSAMINIDASE-H FROM STREPTOMYCES-PLICATUS

In order to identify groups essential for the activity of endo-beta-N- acetylglucosaminidase H (Endo H), all 8 glutamate residues, all 19 asp artates, and both tryptophans were individually substituted with gluta mines, asparagines, and phenylalanines, respectively, by oligonucleoti de site-directed mutagenesis. Only variants D170N, D172N, and E174Q we re found to have specific activities significantly less than wild-type Endo H. Another variant, D173N, did not produce detectable amounts of protein. Wild-type enzyme was found to have a bell-shaped pH activity profile, which was retained in the essential aspartate mutants, but E 174Q lost the basic pH limb of the curve, indicating that E174 is good candidate for the proton donating group necessary for catalysis. The general base needed for activity could not be unambiguously identified ; although, of the essential aspartates, D172 is the only one conserve d in other related glucosidases. (C) 1994 Academic Press, Inc.