Kinetic properties and metal content of the metallo-beta-lactamase CcrA harboring selective amino acid substitutions.
The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations.
Yang
Y
Y
Wyeth-Ayerst Research, Pearl River, New York 10965, USA.
Keeney
D
D
Tang
X
X
Canfield
N
N
Rasmussen
B A
BA
D000596:Amino Acids / Q000235:genetics; D001426:Bacterial Proteins; D002509:Cephaloridine / Q000737:chemistry; D015378:Imipenem / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D010406:Penicillins / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017384:Sequence Deletion / Q000235:genetics; D015032:Zinc / Q000032:analysis; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics; D047090:beta-Lactams / Q000737:chemistry
D000596:Amino Acids; D001426:Bacterial Proteins; D008667:Metalloproteins; D010406:Penicillins; D047090:beta-Lactams; D015378:Imipenem; D001618:beta-Lactamases; C063912:carbapenemase; D015032:Zinc; D002509:Cephaloridine