Escherichia coli outer membrane phospholipase A: role of two serines in enzymatic activity.
In the outer membrane phospholipase A (OMPLA) of Escherichia coli, Ser144 has previously been identified by chemical modification as the active site serine residue. In a specific OMPLA-negative mutant strain, the pldA gene coding for OMPLA was shown to differ from the wild-type gene by a single point mutation, resulting in the substitution of Ser152 by phenylalanine. The role in catalysis of these two serine residues in OMPLA was investigated by site-directed mutagenesis. Ser144 and Ser152 were replaced one at the time by either alanine, valine, phenylalanine, threonine, or cysteine. Ser152 was furthermore replaced by asparagine. Replacement of Ser144 by cysteine resulted in 1% residual activity, whereas the other substitutions at this position yielded virtually inactive enzymes. Substitution of Ser 152 by threonine or asparagine resulted in 40% and 2% residual activity respectively, whereas all other substitutions at this position resulted in the loss of enzymatic activity. We propose that Ser144 is the nucleophile in catalysis, and that Ser152 is involved in hydrogen bonding either to the catalytic triad or in the oxyanion hole.
Brok
R G
RG
Department of Enzymology and Protein Engineering, Utrecht University, The Netherlands.
Belandia
I U
IU
Dekker
N
N
Tommassen
J
J
Verheij
H M
HM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine
D017931:DNA Primers; D011994:Recombinant Proteins; D012694:Serine; D010741:Phospholipases A