David J. Vocadlo

Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The ‘Phillips’ mechanism is widely held as the paradigm for the catalytic mechanism of β-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining β-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for allxa0retaining β-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migrationxa0of C1 along the reaction coordinate.

Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state.

SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-β-hexosaminides. Accumulating evidence suggests that the hydrolytic mechanism involves substrate-assisted catalysis wherein the 2-acetamido substituent acts as a nucleophile to form an oxazolinium ion intermediate. The role of a conserved aspartate residue (D313) in the active site ofSpHex was investigated through kinetic and structural analyses of two variant enzymes, D313A and D313N. Three-dimensional structures of the wild-type and variant enzymes in product complexes with N-acetyl-d-glucosamine revealed substantial differences. In the D313A variant the 2-acetamido group was found in two conformations of which only one is able to aid in catalysis through anchimeric assistance. The mutation D313N results in a steric clash in the active site between Asn-313 and the 2-acetamido group preventing the 2-acetamido group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the insensitivity of this variant to chemical rescue. By comparison, the D313A mutation results in a shift in a shift in the pH optimum and a modest decrease in activity that can be rescued by using azide as an exogenous nucleophile. These structural and kinetic data provide evidence that Asp-313 stabilizes the transition states flanking the oxazoline intermediate and also assists to correctly orient the 2-acetamido group for catalysis. Based on analogous conserved residues in the family 18 chitinases and family 56 hyaluronidases, the roles played by the Asp-313 residue is likely general for all hexosaminidases using a mechanism involving substrate-assisted catalysis.

Characterization of a β‐N‐acetylhexosaminidase and a β‐N‐acetylglucosaminidase/β‐glucosidase from Cellulomonas fimi

The Gram‐positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular β‐N‐acetylglucosaminidases, a family 20 β‐N‐acetylhexosaminidase (Hex20), and a novel family 3‐β‐N‐acetylglucosaminidase/β‐glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: kcat/Km values at 25u2003°C were 0.066u2003s−1·mm−1 and 0.076u2003s−1·mm−1 for 4′‐nitrophenyl β‐N‐acetyl‐d‐glucosaminide and 4′‐nitrophenyl β‐d‐glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between β‐N‐acetylglucosaminidases and β‐glucosidases of family 3. Reaction by a double‐displacement mechanism was confirmed for Nag3 through the identification of a glycosyl–enzyme species trapped with the slow substrate 2′,4′‐dinitrophenyl 2‐deoxy‐2‐fluoro‐β‐d‐glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave β‐glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for the d‐glucosyl/d‐galactosyl configuration of the glycone: Km and kcat values were 53u2003µm and 482.3u2003s−1 for 4′‐nitrophenyl β‐N‐acetyl‐d‐glucosaminide and 66u2003µm and 129.1u2003s−1 for 4′‐nitrophenyl β‐N‐acetyl‐d‐galactosaminide.