COMBINATORIAL MANIPULATION OF 3 KEY ACTIVE-SITE RESIDUES IN GLYCINAMIDE RIBONUCLEOTIDE TRANSFORMYLASE

The enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2) has previously been shown to have three key polar active site residues imp ortant for catalysis: N106, H108 and D144, Mutations of any of these t hree residues lead to substantially decreased catalytic activity, alth ough none of them are completely irreplaceable. In order to determine whether any alternative arrangement of amino acids at these three posi tions could lead to an active protein, all three of these residues wer e simultaneously subjected to saturation site-directed mutagenesis. Th e resulting combinatorial library of mutant genes was screened for tho se encoding active proteins using functional complementation, Glycinam ide ribonucleotide transformylase was found to be capable of toleratin g no more than one mutation amongst these key residues, since the only proteins found to be sufficiently active to allow growth of auxotroph ic cells on selective media were the wild-type and enzymes containing a single mutation to one of these residues. It seems likely that no en zymes 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.