Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.
Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.
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ROB-1 的位点饱和诱变和三维建模定义了 Ser130 在 A 类 β-内酰胺酶中的底物结合作用。
DOI：
10.1093/protein/5.7.693
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Levesque,RC
中科院分区：
文献类型：
--
作者：
Juteau,JM;Billings,E;Knox,JR;Levesque,RC
对 A 类 ROB-1β-内酰胺酶保守的 Ser130 进行位点饱和诱变，该酶位于抗生素结合位点的中心，可以参与蛋白质-蛋白质和蛋白质-底物氢键结合。突变 Thr130 产生β-内酰胺酶，可水解青霉素和头孢菌素，但对氨苄西林和头孢氨苄的亲和力 (Km) 降低 3 倍，对氨苄青霉素的水解 (Vmax 活性) 降低 30 倍。相反，头孢氨苄的水解活性与 Thr130 突变的野生型相似。突变 Gly130 产生β-内酰胺酶仅水解青霉素，对这些化合物的亲和力和水解活性比野生型低约 15 倍，但对头孢菌素没有可检测到的活性，产生的酶只能以低速率水解青霉素，这是根据同源芽孢杆菌的精制 2 Å X 射线结构进行的。氨苄青霉素和头孢氨苄被对接至活性位点，并且仅当 Ser70 为阴离子并且 Glu166 为中性时，对接是稳定的，计算了与 Ser、Thr、Gly 和 Ala130 酶的完全水合预酰化复合物的催化数据。模型证实，Ser 130 在青霉素的结合和水解中具有结构和功能作用，这种高度保守的残基还通过氢与头孢菌素的羧酸基团的结合比青霉素更紧密地发挥底物特异性作用。
Site-saturation mutagenesis was performed on the class A ROB-1β-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein–protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a β-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Kmfor ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmaxactivity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a β-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds ˜ 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 Å X-ray structure of the homologousBacillus licheniformisβ-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser 130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.
神经科学中的细胞培养
DOI：
--
发表时间：
2011
期刊：
Current Topics in Neurobiology
影响因子：
--
作者：
J. Bottenstein;G. Sato
通讯作者：
G. Sato
用于放射免疫测定的最大比活性的 125I 单标记 3,5,3-三碘甲状腺原氨酸和甲状腺素的合成。
影响因子：
2.1
作者：
J. Weeke;H. Orskov
通讯作者：
H. Orskov