Purification and characterization of metallo-beta-lactamase from Serratia marcescens.
Carbapenem-hydrolyzing beta-lactamase from Serratia marcescens FHSM4055 was purified 926-fold by means of carboxylmethyl Sephadex C-50, Sephacryl S-200, and Mono S column chromatography. The molecular weight was 30,000 by SDS-PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p-chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo-beta-lactamase and that the SH-group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (kcat/Km) showed that the enzyme hydrolyzed various beta-lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino- and imino-groups, ceftazidime with a carboxypropyloxyimino-group, and cefclidin with a carbamoylquinuclidine-group were poor substrates among the beta-lactam antibiotics other than the monobactams tested. The plots of the turnover number (kcat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the 'tail' on the acid side (pK1, 5.9; pK2, 9.0; pK3, 10.8), whereas those of kcat/Km gave a bell-shaped curve (pK1, 5.8; pK2, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Marumo
K
K
Department of Clinical Pathology, Showa University Fujigaoka Hospital, Kanagawa, Japan.
Takeda
A
A
Nakamura
Y
Y
Nakaya
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D015780:Carbapenems / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D012706:Serratia marcescens / Q000201:enzymology*; D013379:Substrate Specificity; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D015780:Carbapenems; D002413:Cations, Divalent; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases