Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli.
Penicillin-binding proteins (PBPs) and beta-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced gamma-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two gamma-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Weilie
W
Zhang
Qicun
Q
Shi
Samy O
SO
Meroueh
Sergei B
SB
Vakulenko
Shahriar
S
Mobashery
D000215:Acylation; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D010457:Peptidoglycan; D011485:Protein Binding; D017494:Proton Pumps; D001618:beta-Lactamases / Q000737:chemistry*
D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D017494:Proton Pumps; C000204:S-2-aminoethyl cysteine; D001618:beta-Lactamases; D008239:Lysine; D003545:Cysteine