Probing the non-proline cis peptide bond in beta-lactamase from Staphylococcus aureus PC1 by the replacement Asn136 --> Ala.
A non-proline cis peptide is present between Glu166 and Ile167 in the active site of beta-lactamase from Staphylococcus aureus PC1. To examine the role of the interaction between the side chain of Asn136 and the main chain of Glu166, the site-directed mutant N136A was produced. The enzyme shows no measurable hydrolytic activity toward a variety of penicillins or cephalosporins except for the chromogenic cephalosporin, nitrocefin. For nitrocefin, the progress curve exhibits a fast burst with a stoichiometry of 1 mol of degraded substrate per mole of enzyme followed by a slow phase with a hydrolysis rate that is reduced by approximately 700-fold compared with that of the wild-type enzyme. Thus, the mutant enzyme is deacylation defective. Monitoring the hydrolysis of nitrocefin after preincubation with a number of beta-lactam compounds shows that cephalosporins form stable acyl complexes with the enzyme, whereas penicillins do not. The molecular weight of the mutant was determined by electrospray mass spectrometry, and the presence of the stable acyl enzyme adducts with cephaloridine and cefotaxime was confirmed by both electrospray and MALDI mass spectrometry. Therefore, in addition to impairing deacylation, the acylation machinery has been altered compared with the wild-type enzyme to act on cephalosporins and not on penicillins. Urea denaturation and thermal unfolding studies show that the N136A mutant enzyme is less stable than the wild-type enzyme. However, stability against chemical denaturation of the mutant enzyme is enhanced in the presence of cephaloridine beyond the stability of the wild-type protein. This is attributed to accumulation of favorable interactions between the cephaloridine and the protein, which play a role in the folded state and not in the unfolded state.
Banerjee
S
S
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Shigematsu
N
N
Pannell
L K
LK
Ruvinov
S
S
Orban
J
J
Schwarz
F
F
Herzberg
O
O
D000409:Alanine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D001618:beta-Lactamases; D000409:Alanine