Endo-β-N-acetylglucosaminidase H hydrolyzes
the β-(1–4)-glycosidic link of the
N,N′-diacetylchitobiose
core of high-mannose and hybrid asparagine-linked oligosaccharides.
Seven mutants of the active site residues, Asp130 and Glu132,
have been prepared, assayed, and crystallized. They include
single site mutants of each residue to the corresponding
amide, to Ala and to the alternate acidic residue, and
to the double amide mutant. The mutants of Asp130 are more
active than the corresponding Glu132 mutants, consistent
with the assignment of the latter residue as the primary
catalytic residue. The amide mutants are more active than
the alternate acidic residue mutants, which in turn are
more active than the Ala mutants. The structures of the
Asn mutant of Asp130 and the double mutant are very similar
to that of the wild-type enzyme. Several residues surrounding
the mutated residues, including some that form part of
the core of the β-barrel and especially Tyr168 and
Tyr244, adopt a very different conformation in the structures
of the other two mutants of Asp130 and in the Asp mutant
of Glu132. The results show that the residues in the upper
layers of the β-barrel can organize into two very distinct
packing arrangements that depend on subtle electrostatic
and steric differences and that greatly affect the geometry
of the substrate-binding cleft. Consequently, the relative
activities of several of the mutants are defined by structural
changes, leading to impaired substrate binding, in addition
to changes in functionality.