Mobashery S, Johnston M
Department of Chemistry, Searle Chemistry Laboratory, University of Chicago, Illinois 60637.
Biochemistry. 1987 Sep 8;26(18):5878-84. doi: 10.1021/bi00392a045.
The reactions of a set of amino acid and peptidyl C10-esters of deacetylcephalothin (1-5) have been examined with purified enzymes in vitro. Each of the compounds examined is a substrate for the Escherichia coli TEM-2 beta-lactamase, and enzyme-catalyzed hydrolysis of the lactam bond gives release of an amino acid or a peptidyl fragment from a cephem nucleus. 7 beta-(2-Thienylacetamido)-3-[[(beta-chloro-L-alanyl)oxy]methyl]-3- cephem-4-carboxylate (4) gives time-dependent inactivation of E. coli JSR-O alanine racemase in a process that requires beta-lactamase for the initial liberation of beta-chloro-L-alanine from the cephalosporin. Alanine racemase is similarly inactivated by 7 beta-(2-thienylacetamido)-3-[[[(beta-chloro-L-alanyl)-beta-chloro- L- alanyl]oxy]methyl]-3-cephem-4-carboxylate (1), but this inhibition requires the sequential action of both beta-lactamase and alanine aminopeptidase. Analysis of the enzymatic transformations of 7 beta-(2-thienylacetamido)-3-[[[(beta-chloro-L-alanyl)-L- alanyl]oxy]methyl]-3-cephem-4-carboxylate (3), monitored by high-field 1H NMR, reveals that (1) beta-lactamase releases the dipeptide beta-chloro-L-alanyl-L-alanine from 3 and (2) leucine aminopeptidase effects stoichiometric hydrolysis of the dipeptide to beta-chloro-L-alanine and L-alanine. These biochemical findings are discussed with reference to the mechanism of antibacterial action of 1 against beta-lactamase-producing, penicillin-resistant microorganisms [Mobashery, S., Lerner, S. A., & Johnston, M. (1986) J. Am. Chem. Soc. 108, 1685].
已在体外使用纯化的酶检测了一组脱乙酰头孢菌素（1 - 5）的氨基酸和肽基C10酯的反应。所检测的每种化合物都是大肠杆菌TEM - 2β - 内酰胺酶的底物，酶催化的内酰胺键水解会从头孢烯核释放出氨基酸或肽基片段。7β - （2 - 噻吩基乙酰胺基） - 3 - [[（β - 氯 - L - 丙氨酰基）氧基]甲基] - 3 - 头孢烯 - 4 - 羧酸酯（4）在一个需要β - 内酰胺酶从头孢菌素最初释放β - 氯 - L - 丙氨酸的过程中，会使大肠杆菌JSR - O丙氨酸消旋酶发生时间依赖性失活。丙氨酸消旋酶同样会被7β - （2 - 噻吩基乙酰胺基） - 3 - [[[（β - 氯 - L - 丙氨酰基） - β - 氯 - L - 丙氨酰基]氧基]甲基] - 3 - 头孢烯 - 4 - 羧酸酯（1）失活，但这种抑制需要β - 内酰胺酶和丙氨酸氨肽酶的顺序作用。通过高场1H NMR监测7β - （2 - 噻吩基乙酰胺基） - 3 - [[[（β - 氯 - L - 丙氨酰基） - L - 丙氨酰基]氧基]甲基] - 3 - 头孢烯 - 4 - 羧酸酯（3）的酶促转化分析表明：（1）β - 内酰胺酶从3中释放出二肽β - 氯 - L - 丙氨酰基 - L - 丙氨酸；（2）亮氨酸氨肽酶将二肽化学计量水解为β - 氯 - L - 丙氨酸和L - 丙氨酸。结合1对产生β - 内酰胺酶的耐青霉素微生物的抗菌作用机制，对这些生化发现进行了讨论[莫巴舍里，S.，勒纳，S. A.，& 约翰斯顿，M.（1986年）《美国化学会志》108，1685]。
