Synergistic contributions of asparagine 46 and aspartate 52 to the catalytic mechanism of chicken egg white lysozyme.
The X-ray structure of a chicken egg white lysozyme (ChEWL) complex with a peptidoglycan-derived inhibitor suggests that interactions of Asn46 and Asp52 with the D-subsite N-acetylmuramic acid residue help to distort that pyranose ring into the reactive half-chair conformation and that a hydrogen bond is formed between Asn46 and Asp52 [Strynadka, N. C. J., & James, M. N. G. (1991) J. Mol. Biol. 220, 401-424]. These hypotheses were investigated through the D52A, N46A, and D52A/N46A mutants of ChEWL. The Michaelis constants of the D52A and D52A/N46A ChEWL complexes with Micrococcus luteus cells are 3- and 4-fold higher, respectively, than the wild-type KM; the corresponding kcat values are 25- and 50-fold lower, respectively, than the wild-type kcat. These results support the proposal of Strynadka and James. The velocities of reactions catalyzed by the N46A and D52A mutants are approximately equal to each other for all classes of substrate, suggesting that the respective roles of Asn46 and Asp52 in transition state stabilization do not vary. The mutation of either Asn46 or Asp52 to Ala apparently disrupts the interactions of the other (nonmutated) residue with the substrate, supporting the crystallographic evidence of a hydrogen-bond interaction between the two residues. The mutations do not change the values of the dissociation constants of complexes with (carboxymethyl)chitin complexes, suggesting that ground state complexes of ChEWL with chitin-derived substrates differ in conformation from complexes with bacterial peptidoglycans.
Matsumura
I
I
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Kirsch
J F
JF
D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002473:Cell Wall / Q000737:chemistry; D002645:Chickens; D002686:Chitin / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D016982:Micrococcus luteus / Q000737:chemistry; D008958:Models, Molecular; 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
D009838:Oligodeoxyribonucleotides; D002686:Chitin; D001224:Aspartic Acid; D001216:Asparagine; D009113:Muramidase