Catalytic mechanism of active-site serine beta-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad.
The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.
Dubus
A
A
Centre d'Ingénierie des Protéines, Université de Liège, Belgium.
Wilkin
J M
JM
Raquet
X
X
Normark
S
S
Frère
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010406:Penicillins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D012694:Serine; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002511:Cephalosporins; D010406:Penicillins; D012694:Serine; D001618:beta-Lactamases