Tan A K, Fink A L
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
Biochem J. 1992 Jan 1;281 ( Pt 1)(Pt 1):191-6. doi: 10.1042/bj2810191.
Nafcillin was shown to reversibly inhibit beta-lactamase from Staphylococcus aureus PC1 with characteristics indicative of a type A inhibitor [Citri, Samuni & Zyk (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1048-1052]. At nafcillin concentrations above 80 mM, complete inactivation occurred within 200 s. Upon removal of the excess nafcillin the inhibited enzyme was re-activated completely, with a rate constant of 2.0 x 10(-3) s-1 (25 degrees C). The inhibited enzyme was shown to be in the form of a covalent acyl-enzyme intermediate. Digestion by pepsin and trypsin yielded a single nafcillin-labelled peptide fragment which was isolated, sequenced and shown to be: Ala-Tyr-Ala-Ser-Thr-Ser-Lys. This sequence corresponds to the region surrounding the active-site serine residue, Ser-70, indicating that the inhibitor is covalently attached to the same residue as productive substrates.
已证明萘夫西林可可逆地抑制金黄色葡萄球菌PC1的β-内酰胺酶,其特征表明它是一种A型抑制剂[Citri、Samuni和Zyk(1976年),《美国国家科学院院刊》73, 1048 - 1052]。在萘夫西林浓度高于80 mM时,200秒内会发生完全失活。去除过量的萘夫西林后,被抑制的酶会完全重新激活,速率常数为2.0×10⁻³ s⁻¹(25℃)。被抑制的酶显示为共价酰基酶中间体的形式。用胃蛋白酶和胰蛋白酶消化产生了一个单一的萘夫西林标记的肽片段,该片段被分离、测序并显示为:Ala-Tyr-Ala-Ser-Thr-Ser-Lys。该序列对应于活性位点丝氨酸残基Ser-70周围的区域,表明抑制剂与有效底物共价连接到同一个残基上。