Fukagawa Y, Takei T, Ishikura T
Biochem J. 1980 Jan 1;185(1):177-85. doi: 10.1042/bj1850177.
By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.
通过使用一种新的计算机辅助紫外分光光度测定法,重新研究了地衣芽孢杆菌749/Cβ-内酰胺酶催化反应的动力学参数,并以苄青霉素为底物研究了新型β-内酰胺抗生素化合物PS-5对该酶的抑制模式。(1) 以苄青霉素为底物,详细研究了测定Km和V的基本测定条件。在0.1M磷酸钠/钾缓冲液(pH6.8)中,30℃下,苄青霉素初始底物浓度高于0.7mM很可能导致底物抑制。在初始浓度为1.96至0.07mM时,该酶对苄青霉素的Km值经计算为97 - 108μM。(2) 发现该酶对6-氨基青霉烷酸、氨苄青霉素和头孢菌素的Km值分别为25、154 - 161和144 - 161μM。(3) 化合物PS-5实际上未被地衣芽孢杆菌749/Cβ-内酰胺酶攻击。(4) 化合物PS-5会降低该酶的活性,降低程度取决于孵育时间和抑制剂浓度。化合物PS-5使该酶失活的速率符合一级动力学。(5) 在附录中,设计了一种新的计算机辅助紫外分光光度酶测定法,通过积分米氏方程分析单一的时间 - 吸光度反应进程曲线,用于准确、精确地测定β-内酰胺酶的动力学常数。为了将吸光度读数转换为摩尔底物浓度,将与反应时间无关的初始或最终吸光度读数用作计算基础。在计算Km和V时,采用了三种系统的数据组合方法对反应进程曲线进行更精细的分析。名为YF6TAIM的计算机程序列表可应作者要求获取,或按照《生物化学杂志》(1978年)169, 5中所示条款从英国西约克郡韦瑟比波士顿斯帕的英国国家图书馆出借部获取补充出版物SUP 50100(12页)。