Cysteine 265 is in the active site of, but is not essential for catalysis by tRNA-guanine transglycosylase (TGT) from Escherichia coli.
Cysteine 265 is in the active site of, but is not essential for catalysis by tRNA-guanine transglycosylase (TGT) from Escherichia coli.
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半胱氨酸 265 位于大肠杆菌 tRNA-鸟嘌呤转糖基酶 (TGT) 的活性位点，但不是催化所必需的。
DOI：
10.1023/a:1026334726357
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Chong,S
中科院分区：
文献类型：
--
作者：
Garcia,GA;Chong,S
位点定向诱变和x射线吸收光谱研究已经表明，trna -鸟嘌呤转糖基酶（TGT）从大肠杆菌中获得锌金属蛋白并鉴定出锌的酶配体[Chonget al.(1995)，生物化学34,3694-3701；Biochemistry35 Garciaet。(1966),3133 - 3139]。在这些研究中，发现一个突变体TGT （C265A）表现出明显较低的特异性活性，但未发现与锌位点有关。本报告表明，TGT可通过巯基试剂（如DTNB、MMTS和n -乙基马来酰亚胺）灭活。此外，这种失活被证明是由于半胱氨酸265的修饰。然而，突变体TGT （C265A）和TGT （C265S）的动力学参数表明，该残基在TGT反应中没有发挥关键作用。我们得出结论，半胱氨酸265位于TGT的活性位点，但没有发挥关键的催化作用。最近对活动单胞菌TGT的x射线晶体结构的测定支持了这一结论[Romieret al.(1966),EMBO J.15, 2850-2857]，结果表明，半胱氨酸265对应的残基远离假定的催化位点，但位于酶表面与tRNA结合区域的中间。
Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) fromEscherichia coliis a zinc metalloprotein and identified the enzymic ligands to the zinc [Chonget al.(1995),Biochemistry34,3694–3701; Garciaet al.(1966),Biochemistry35,3133–3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents (e.g.,DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT fromZymomonas mobilis[Romieret al.(1966),EMBO J.15,2850–2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA.