Study on function of the active-site of cephalosporinase type beta-lactamase
| Project/Area Number |
01571194
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Chiba University |
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Principal Investigator |
SAWAI Tetsuo Chiba Univ. Fac. of Pharm. Sci., Professor, 薬学部, 教授 (70009174)
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| Co-Investigator(Kenkyū-buntansha) |
TSUKAMOTO Kikuo Chiba Univ. Fac. of Pharm. Sci., Assistant, 薬学部, 助手 (20183478)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1989: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | beta-Lactamase / Cephalosporinase / Active-site / beta-Lactam Antibiotics / Drug Resistance / Site-directed Mutagenesis / Gram-negative Bacteria / β-ラクタマ-ゼ / β-ラクタム抗生物質 |
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| Research Abstract |
This study was carried out for elucidating the molecular structure of the active-site of a cephalosporinase type bata-lactamase and the function of amino acid residues located in the active-site. The results of this study will contribute to the improvelopments in bata-lactam antibiotics and specific inhibitors for beta-lactamases. The beta-lactamase gene was cloned from the chromosomal DNA of Citrobacter Freundii to a vector plasmid and the nucleotide sequence was completely determined. From the nuclotide sequence, the amino acid sequence of the mature enzyme was deduced (361 amino acids). Ser-64 was also confirmed to be the catalytic site. On the basis of the comparison between the amino acid sequence of the C.freeundii beta-lactamase and those of known beta-lactamases, the amino acid residues constructing the active-site space were speculated. Those residues were expected to participate in the catalytic action or to determine the substrate spectrum. Lys-67, Tyr-150 and Lys-315 which were assumed to be situated in positions close to Ser-64 in the three-dimensional structure of the enzyme protein, were converted into other amino acids by site-directed mutagenesis, respectively. The kinetic investigations of these mutant enzymes revealed the contribution of the individual residues to the enzyme activity. Asp-217 and Glu-219 were assumed to be located at the rim of the active-site hollow, and be unable to interact directly with the substrate in the active-site hollow. However, we found an interesting fact that the substrate spectrum of the mutant enzymes were extended to oxyimino cephalosporins. On the other hand, the mutant enzymes showed a lower activity to cephamycins.
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Report
(3 results)
Research Products
(12 results)
