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首页> 外文期刊>Biochemistry >Investigation of the ATP binding site of Escherichia coli aminoimidazole ribonucleotide synthetase using affinity labeling and site-directed mutagenesis.
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Investigation of the ATP binding site of Escherichia coli aminoimidazole ribonucleotide synthetase using affinity labeling and site-directed mutagenesis.

机译：使用亲和标记和定点诱变研究大肠杆菌氨基咪唑核糖核苷酸合成酶的ATP结合位点。

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摘要

Aminoimidazole ribonucleotide (AIR) synthetase (PurM) catalyzes the conversion of formylglycinamide ribonucleotide (FGAM) and ATP to AIR, ADP, and P(i), the fifth step in de novo purine biosynthesis. The ATP binding domain of the E. coli enzyme has been investigated using the affinity label [(14)C]-p-fluorosulfonylbenzoyl adenosine (FSBA). This compound results in time-dependent inactivation of the enzyme which is accelerated by the presence of FGAM, and gives a K(i) = 25 microM and a k(inact) = 5.6 x 10(-)(2) min(-)(1). The inactivation is inhibited by ADP and is stoichiometric with respect to AIR synthetase. After trypsin digestion of the labeled enzyme, a single labeled peptide has been isolated, I-X-G-V-V-K, where X is Lys27 modified by FSBA. Site-directed mutants of AIR synthetase were prepared in which this Lys27 was replaced with a Gln, a Leu, and an Arg and the kinetic parameters of the mutant proteins were measured. All three mutants gave k(cat)s similar to the wild-type enzyme and K(m)s for ATP less than that determined for the wild-type enzyme. Efforts to inactivate the chicken liver trifunctional AIR synthetase with FSBA were unsuccessful, despite the presence of a Lys27 equivalent. The role of Lys27 in ATP binding appears to be associated with the methylene linker rather than its epsilon-amino group. The specific labeling of the active site by FSBA has helped to define the active site in the recently determined structure of AIR synthetase [Li, C., Kappock, T. J., Stubbe, J., Weaver, T. M., and Ealick, S. E. (1999) Structure (in press)], and suggests additional flexibility in the ATP binding region.
机译：氨基咪唑核糖核苷酸（AIR）合成酶（PurM）催化甲酰甘氨酰胺核糖核苷酸（FGAM）和ATP转化为AIR，ADP和P（i），这是从头进行嘌呤生物合成的第五步。已使用亲和标记[（14）C]-对氟磺酰基苯甲酰基腺苷（FSBA）研究了大肠杆菌酶的ATP结合域。该化合物导致酶的时间依赖性失活，该失活因FGAM的存在而加速，并给出K（i）= 25 microM和ak（inact）= 5.6 x 10（-）（2）min（-）（ 1）。失活被ADP抑制，并且相对于AIR合成酶是化学计量的。用胰蛋白酶消化标记的酶后，已分离出单个标记的肽I-X-G-V-V-K，其中X是被FSBA修饰的Lys27。制备了AIR合成酶的定点突变体，其中该Lys27被Gln，Leu和Arg取代，并测量了该突变体蛋白的动力学参数。所有这三个突变体的k（cat）与野生型酶相似，而ATP的K（m）小于野生型酶的k（cat）。尽管存在Lys27等效物，但用FSBA灭活鸡肝三功能AIR合酶的努力仍未成功。 Lys27在ATP结合中的作用似乎与亚甲基接头而不是其ε-氨基有关。 FSBA对活性位点的特殊标记有助于在最近确定的AIR合酶结构中定义活性位点[Li，C.，Kappock，TJ，Stubbe，J.，Weaver，TM和Ealick，SE（1999）结构（印刷中）]，并建议在ATP结合区域具有额外的灵活性。

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