Warren M S, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Protein Eng. 1997 Jan;10(1):63-8. doi: 10.1093/protein/10.1.63.
The enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2) has previously been shown to have three key polar active site residues important for catalysis: N106, H108 and D144. Mutations of any of these three residues lead to substantially decreased catalytic activity, although none of them are completely irreplaceable. In order to determine whether any alternative arrangement of amino acids at these three positions could lead to an active protein, all three of these residues were simultaneously subjected to saturation site-directed mutagenesis. The resulting combinatorial library of mutant genes was screened for those encoding active proteins using functional complementation. Glycinamide ribonucleotide transformylase was found to be capable of tolerating no more than one mutation amongst these key residues, since the only proteins found to be sufficiently active to allow growth of auxotrophic cells on selective media were the wild-type and enzymes containing a single mutation to one of these residues. It seems likely that no enzymes containing two or more mutations of these three residues possess significant catalytic activity. The combinatorial approach used could prove to be quite useful in protein engineering and protein evolution experiments.
甘氨酰胺核糖核苷酸转甲酰基酶（EC 2.1.2.2）先前已被证明具有三个对催化作用至关重要的关键极性活性位点残基：N106、H108和D144。这三个残基中的任何一个发生突变都会导致催化活性大幅下降，尽管它们都不是完全不可替代的。为了确定这三个位置上氨基酸的任何其他排列是否会产生有活性的蛋白质，对这三个残基同时进行了饱和定点诱变。使用功能互补筛选所得的突变基因组合文库，以寻找编码有活性蛋白质的基因。发现甘氨酰胺核糖核苷酸转甲酰基酶在这些关键残基中最多只能耐受一个突变，因为在选择性培养基上发现的唯一具有足够活性以允许营养缺陷型细胞生长的蛋白质是野生型以及含有这三个残基之一的单个突变的酶。似乎不太可能存在含有这三个残基的两个或更多突变的酶具有显著的催化活性。所使用的组合方法在蛋白质工程和蛋白质进化实验中可能会被证明非常有用。
