Is aspartate 52 essential for catalysis by chicken egg white lysozyme? The role of natural substrate-assisted hydrolysis.
The chicken and goose egg white lysozymes (ChEWL and GoEWL) are homologues, but differ in substrate specificity. ChEWL catalyzes the hydrolysis of the glycosidic bonds of bacterial peptidoglycans and chitin-derived substrates, while GoEWL is specific for bacterial peptidoglycans. The active-site aspartate 52 residue of ChEWL, which is postulated to stabilize the oxocarbenium ion intermediate, has no counterpart in GoEWL. The substrate specificity of the D52A ChEWL mutant was compared with those of wild-type ChEWL and GoEWL. D52A ChEWL retains approximately 4% of the wild-type catalytic activity in reactions with three different bacterial cell suspensions. Asp52 therefore is not essential to the catalytic mechanism, accounting for only a 2 kcal/mol decrease in delta G++. The function of Asp52 in D52A ChEWL- and GoEWL-catalyzed cleavage of (carboxymethyl)chitin may be partially fulfilled by an appropriately positioned carboxyl group on the substrate (substrate-assisted catalysis). D52A ChEWL and GoEWL, unlike wild-type ChEWL, exhibit biphasic kinetics in the clearing of Micrococcus luteus cell suspensions, suggesting preferences for subsets of the linkages in the M. luteus peptidoglycan. These subsets do not exist in the peptidoglycans of Escherichia coli or Sarcina lutea, since neither D52A ChEWL nor GoEWL exhibits initial bursts in reactions with suspensions of these bacteria. We propose that substrate-assisted catalysis occurs in reactions of D52A ChEWL and GoEWL with M. luteus peptidoglycans, with the glycine carboxyl group of un-cross-linked peptides attached to N-acetylmuramic acid partially substituting the function of the missing Asp52.
Matsumura
I
I
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Kirsch
J F
JF
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002384:Catalysis; D002645:Chickens; D005260:Female; D005777:Geese; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010063:Ovum / Q000201:enzymology; D017354:Point Mutation; D013379:Substrate Specificity; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D001224:Aspartic Acid; D009113:Muramidase