Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.

Abstract

The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits =5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% ± 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes are 4.1 ,LM (D52N) and 13.4 pzM (E35Q) vs. 8.6 ,uM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of =a5 mg per liter of culture by high-salt elution from the cell walls.Chicken egg white lysozyme (CEWL; EC 3.2.1.17) has a distinguished history in the field of mechanistic enzymology. It was the first enzyme for which an atomic resolution x-ray structure was published (1), and the essence of the presently accepted catalytic mechanism was proposed almost exclusively from the structural information (2). The enzyme has additionally served as an important paradigm for studies in (i) the physical biochemistry of polydentate liganding, whereby the individual contributions to the several binding subsites have been separately evaluated (3); (ii) molecular evolution investigations (4, 5); and (iii) immunochemical studies (6). The enzyme is thus an attractive candidate for modification by site-directed mutagenesis, because the availability of specifically mutated constructs will have the potential to contribute significantly to all of the above investigations, as well as to studies on protein folding and stability. We report here the relatively high-efficiency expression of Strains. Plasmids were propagated in HB101 grown in LB medium (7). M13mpl8 subclones were grown in JM103 and in RZ1032 for mutagenesis (8). Saccharomyces cerevisiae strain GRF180 is leu2-3,-J12, ura3-52, his4-580, can (cir") and was derived from GRF18 by curing this strain of its endogenous 2-,um circle (9, 10). Escherichia coli and S. cerevisiae transformations were carried out as described (11,12).Plasmids. Plasmid pAB24 is a yeast-E. coli shuttle vector derived from pBR322 and pJDB219 (13). It contains the complete 2-gm circle and the LEU2-D and URA3 genes for selection and replication in yeast and pBR322 sequences and sequences for selection and replication in E. coli (14). Plasmid pAGAP1 is a derivative of pPGAP1 (15) that has the GAPDH-491 promoter replaced with a hybrid ADH2-GAPDH promoter fusion (14). Plasmid pLS1023 containing a CEWL cDNA clone (16) was the generous gift of G. Schutz (University of Cologne). DNA sequencing revealed two discrepancies between pLS1023 and the published sequence, which were repaired by site-directed mutagenesis.Site-Directed Mutagenesis of the Active Site. Two 21-base synthetic primers ...

Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories

“…Site-directed mutagenesis of the hen egg-white lysozyme gene was performed according to Malcolm et al (1989) with modifications described by Shih et al (1993) and Matsumura and Kirsch (1996). Expression and purification were carried out according to KamMorgan et al (1993) and Matsumura and Kirsch (1996).…”

Atomic structure at 2.5 Å resolution of uridine phosphorylase from E. coli as refined in the monoclinic crystal lattice

“…It was putative active site, the titration of at least one of them with demonstrated that substitutions of this residue for homoserine silver or mercury ions leads to a complete loss of the Upase by selective chemical modification [21] or for asparagine by activity. It was shown that Cys 136 may be selectively modified site-directed mutagenesis [22] led to significant enzyme inactiwith 2,4,6-trinitrobenzene sulfonate, the modification being acvation (90-95%) without sufficient changes in the substrate companied by enzyme inactivation. Phosphate addition would affinity.…”

Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics

“…Biochemical investigation of the HEWL.Fab-IO system are facilitated by the heterologous expression of HEWL (Malcolm et al, 1989). The antibody partially occludes the HEWL active site causing the HEWL.Fab-10 complex to be nearly devoid of lysozyme activity.…”