Electron spin echo envelope modulation studies of the Cu(II)-substituted derivative of isopenicillin N synthase: a structural and spectroscopic model.
Electron spin echo envelope modulation studies of the Cu(II)-substituted derivative of isopenicillin N synthase: a structural and spectroscopic model.
复制标题
异青霉素 N 合酶 Cu(II) 取代衍生物的电子自旋回波包络调制研究：结构和光谱模型。
DOI：
10.1021/bi00112a010
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Chen,VJ
中科院分区：
文献类型：
--
作者：
Jiang,F;Peisach,J;Ming,LJ;QueJr,L;Chen,VJ
材料和方法。根据报道的方法（Kriauciunas等人，1991年）。底物ACV来自Incell（密尔沃基，WI），并且所用的所有其它化学品是可商购的。Fe（II）-重构IPNS的催化活性由ACV在0.1M MOPS，pH 7.1和25 ℃中的初始氧消耗速率测定，和/或通过HPLC直接测量在上述条件下产生的异青霉素N [1单位的IPNS活性是基于在上述条件下消耗（形成）1/nmol O2（异青霉素N）/min定义的]。将当用Fe（II）重构时比活性> 4单位/mg的那些酶制剂合并并通过超滤浓缩，然后储存在液氮中直至使用。通过在所需pH下将Cu（II）缓慢输注到在0.1M MOPS缓冲液中的apo-IPNS中来制备Cu（II）IPNS。首先通过在pD 7.1下用在D20中的0.1M MOPS透析apo-IPNS溶液来制备在D20缓冲液中的酶样品，以使蛋白质中的溶剂可交换质子氘化，然后进行Cu（II）重构。通过在厌氧条件下向Cu（II）IPNS溶液中加入> 1当量的ACV来制备Cu（II）IPNS的底物络合物。通过可见光谱和EPR光谱监测Cu（II）IPNS和Cu（II）IPNS-ACV络合物的形成（Ming等人，1990年）。
Materials and Methods. Isopenicillin N synthase from Cephalosporium acremonium (MW 38 400) was isolated as the apo form from recombinant Escherichia coli according to reported procedures (Kriauciunas et al., 1991). The substrate ACV was from Incell (Milwaukee, WI), and all other chemicals used were commercially available. The catalytic activity of Fe (II)-reconstituted IPNS was determined from the initial rate of dioxygen consumptionwith ACV in 0.1 M MOPS, pH 7.1 and 25 C, and/or by direct measurement by HPLC of the isopenicillin N produced under the above con-ditions [1 unit of IPNS activity is defined on the basis of consumption (formation) of 1/nmol of 02 (isopenicillin N)/min under the above conditions]. Those enzyme preparations with specific activity of> 4 units/mg when reconstituted with Fe (II) were pooled and concentrated by ultrafiltration and then stored in liquid nitrogen until used. Cu (II) IPNS was prepared by slow infusion of Cu (II) into apo-IPNS in 0.1 M MOPS buffer at the desired pH. The enzyme samples in D20 buffer were prepared first by dialyzing the apo-IPNS solution against 0.1 M MOPS in D20 at pD 7.1 to deuterate solvent-exchangeable protons in the protein, followed by Cu (II) reconstitution. The substrate complex of Cu (II) IPNS was prepared by the addition of> 1 equiv of ACV to a Cu (II) IPNS solution under anaerobic conditions. The formation of Cu (II) IPNS and the Cu (II) IPNS-ACV complex was monitored by both visible and EPR spectroscopies (Ming et al., 1990).
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与任意自旋核的超精细相互作用产生的电子自旋回波的调制效应
DOI：
--
发表时间：
1981
期刊：
影响因子：
--
作者：
S. Dikanov;A. Shubin;V. Parmon
通讯作者：
V. Parmon
影响因子：
46.2
作者：
J. Robinson
通讯作者：
J. Robinson
影响因子：
15
作者：
JIANG, F;MCCRACKEN, J;PEISACH, J
通讯作者：
PEISACH, J
半乳糖氧化酶中铜 (II) 位点的脉冲电子顺磁共振研究。
影响因子：
2.9
作者：
D. Kosman;J. Peisach;W. Mims
通讯作者：
W. Mims
异青霉素N合酶金属结合位点的配位化学
影响因子：
4.6
作者：
L. Ming;L. Que;A. Kriauciunas;C. Frolik;V. J. Chen
通讯作者：
V. J. Chen