Identification of an essential glutamic acid residue in beta-lactamase II from Bacillus cereus.
Beta-Lactamase II from Bacillus cereus was readily inactivated by incubation at pH 4.75 with a water-soluble carbodiimide plus a suitable nucleophile. In the early stages of the reaction, 1 equivalent of nucleophile was incorporated/equivalent of enzyme, whereas during the later stages a second equivalent of nucleophile was also incorporated. This latter process correlated with the blocking of the enzyme's single thiol group. Enzyme inactivated in the presence of the coloured nucleophile N-(2,4-dinitrophenyl)ethylenediamine was fragmented by pepsin digestion, and coloured peptides were isolated by gel filtration and h.p.l.c. Two major peptides, representing 52% of the incorporated label, were isolated and sequenced. Both peptides contained the incorporated label on glutamic acid-37, and it is concluded that this latter residue represents a catalytically essential carboxylic residue in beta-lactamase II.
Little
C
C
Emanuel
E L
EL
Gagnon
J
J
Waley
S G
SG
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D002510:Cephalosporinase / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005022:Ethyldimethylaminopropyl Carbodiimide; D005971:Glutamates / Q000032:analysis*; D018698:Glutamic Acid; D008954:Models, Biological; D010446:Peptide Fragments / Q000032:analysis; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
D005971:Glutamates; D010446:Peptide Fragments; D065093:beta-Lactamase Inhibitors; D018698:Glutamic Acid; D002510:Cephalosporinase; D001618:beta-Lactamases; D005022:Ethyldimethylaminopropyl Carbodiimide