PROBING THE ACTIVE SITE OF ENDO H BY MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY. Vibha Rao^1,2, and Patrick Van Roey¹, (1) Wadsworth Center, New York State Dept. of Health, Albany, NY 12201, USA, and (2) Physics Dept., University at Albany, Albany, NY 12222, USA

Endo-(-N-acetylglucosaminidase H, an endoglycosidase secreted by Streptomyces plicatus, hydrolyzes the central ((1-4) glycosidic bond between the core N-acetylglucosamine residues of asparagine-linked oligosaccharides. It requires the following minimum substrate: -Man((1-3)Man((1-6)Man((1-4)GlcNAc((1-4)GlcNAc-Asn, and therefore is highly specific for high mannose and hybrid glycans. The overall fold of Endo H is that of an ((/()₈ -barrel (Rao, V. et al., Structure, 3, 449-457, 1995). Site directed mutagenesis studies have resulted in the identification of Asp130 and Glu132 as catalytic residues. Mutations of Asp130 to Asn (D130N), Glu (D130E) and Ala (D130A) resulted in 0.1 - 1.0% activity of the wild-type enzyme. However, the Glu132 mutants, E132A and E132Q have no detectable activity. Crystal structures of three mutants, D130N, E132A and the double mutant D130N + E132Q, have been determined to 2.1 resolution by molecular replacement methods. Mutants of E132 crystallize in a different crystal form (P2₁) than the wild-type enzyme (P4₃2₁2), apparently resulting from the absence of an intermolecular contact made by Glu132 in the wild-type form. Detailed comparison of the structures shows no notable change in the backbone conformations. The r.m.s. deviations of main chain atoms compared to the wild-type structure are: D130N mutant (space group P4₃2₁2), 0.38 ; E132A (P2₁), 0.54 ; and, D130N + E132Q (P2₁), 0.58 . Side-chains of hydrophobic residues in the area of the active-site vary more in conformation than those of polar residues. Hence, mutagenesis experiments and the crystal structures of the active-site mutants indicate that Glu132 is absolutely essential for the hydrolysis and that Asp130 may not participate directly in the catalysis, but may serve to create a more negative environment around Glu132.