Arg362 and Tyr365 of the botulinum neurotoxin type a light chain are involved in transition state stabilization
Arg362 and Tyr365 of the botulinum neurotoxin type a light chain are involved in transition state stabilization
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DOI：
10.1021/bi0157969
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发表时间：
2002-02-12
期刊：
影响因子：
2.9
通讯作者：
Alves, J
中科院分区：
文献类型：
--
作者：
Binz, T;Bade, S;Alves, J
肉毒杆菌神经毒素A型（BoNT/A）轻链（LC）充当锌内肽酶。该毒素的X射线结构表明，Zn 2+由Zn 2+结合基序HisGluXXHis和Glu（261）的His（222）和His（226）配位，而Glu（223）作为第四配体配位水解所需的水分子。最近对BoNT/B LC及其底物小突触素2的共晶体的分析表明，同源BoNT/A的Arg（362）和Tyr（365）可能直接参与催化。通过定点突变分析了它们的作用和也在活性位点附近发现的Glu（350）的作用。用Phe对Arg（362）的各种替换和对Tyr（365）的替换分别导致k（cat）/K-m值降低79倍和34倍。这些变化是由降低的催化速率（kg）引起的，而不是由基态底物结合的改变引起的，这一点可以通过基本不变的Kd和K-m值来证明。这些突变均不影响LC的总体二级结构或锌含量。这些发现表明Arg（362）的胍基基团和Tyr（365）的羟基基团一起实现过渡态稳定化，如针对嗜热菌蛋白酶提出的，嗜热菌蛋白酶是金属蛋白酶的谷锌蛋白超家族的原型成员。Glu（350）突变显著降低了水解活性，这必须部分归因于活性位点精细结构的改变，如对热诱导变性的敏感性增加和较低的Zn 2+结合亲和力所示。Glu（350）显然占据活性位点的中心位置，并且推测位置为His（222）和Arg（362）。
The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn2+ is coordinated by His(222) and His(226) of the Zn2+ binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K-m values, respectively. These changes were provoked by decreased catalytic rates (kg) and not by alterations of ground state substrate binding as evidenced by largely unchanged K-d and K-m, values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn2+ binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).