Fisher J, Belasco J G, Khosla S, Knowles J R
Biochemistry. 1980 Jun 24;19(13):2895-901. doi: 10.1021/bi00554a012.
The use of cefoxitin, a poor substrate of the RTEM beta-lactamase, has allowed the kinetic and spectroscopic characterization of a covalent acyl-enzyme intermediate in the enzyme-catalyzed reaction. The rate of reappearance of catalytic activity in an enzyme sample diluted from an incubation with cefoxitin is nearly identical with the observed Kcat. Burst kinetics are observed with this substrate, consistent with the rate-limiting deacylation of the cefoxitinoyl-enzyme. That the reaction intermediate involves a covalent link between enzyme and substrate was shown by gel filtration after rapid denaturation of an enzyme-[14C]cefoxitin reaction at the steady state. Fourier transform infrared measurements indicate that the intermediate is an acyl-enzyme involving a hydroxyl group of the beta-lactamase. The evident relationship between the acylation-deacylation sequence of the beta-lactamases and the acylation reaction suffered by the D-Ala-D-Ala-carboxypeptidases is discussed.
头孢西丁是RTEMβ-内酰胺酶的一种劣质底物，其使用使得能够对酶催化反应中的共价酰基酶中间体进行动力学和光谱表征。在与头孢西丁孵育后稀释的酶样品中，催化活性重新出现的速率与观察到的Kcat几乎相同。用这种底物观察到了爆发动力学，这与头孢西丁酰基酶的限速脱酰作用一致。在稳态下对酶-[14C]头孢西丁反应进行快速变性后，通过凝胶过滤表明反应中间体涉及酶与底物之间的共价连接。傅里叶变换红外测量表明，中间体是一种涉及β-内酰胺酶羟基的酰基酶。讨论了β-内酰胺酶的酰化-脱酰序列与D-Ala-D-Ala-羧肽酶所经历的酰化反应之间的明显关系。
