| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
In the clinical field, cephalosporin hydrolysing class C beta-lactamase are of importance because clinical isolates producing class C beta-lactamase show high levels of resistance to third generation oxyimino cephalosporins. To survey functional amino acids of the class C beta-lactamase, a series of mutant gense of the class C beta-lactamase of Citrobacter freundii GN346 were constructed by site-directed mutagenesis. Three conserved residues, i.e., Lys67, Tyr150 and Lys315 were identified to be the catalytic residues because the substitution of these residues to non-conserved amino acids resulted in a dramatic decrease in the enzyme activity. Change of kinetic parameters to favorable substrates, the kinetic analysis of the acyl-enzyme intermediate formation by using ^<14>C-benzylpenicillin as a substrate indicated that these three residues play important role in catalytic mechanism. To estimate the interaction between residues lying in the active site pocket and a favorable beta-lactam substrate, molecular dynamic symulation was performed. This result sugested that Lys67 assists the active site Ser64 in the acylation step, and the other two residues, Tyr150 and Lys315, may involed in the deacylation step.
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