Probing histidine-substrate interactions in tyrosyl-tRNA synthetase using asparagine and glutamine replacements.

We have analyzed the interactions of a histidine residue with a substrate using site-directed mutagenesis. Previous studies on tyrosyl-tRNA synthetase from Bacillus stearothermophilus have shown that a histidine residue (His-48) makes an interaction with ATP, which is improved on mutating Thr-51----Pro-51. We find on replacing His-48 in wild-type enzyme with either asparagine or glutamine that Asn-48 is equally as good as His-48 but His-48----Gln-48 leads to a far lower activity. The side chain of an asparagine residue may be superimposed on that of a histidine so that the amide-NH2 group of asparagine occupies the same position as the pi-N of histidine, whereas the equivalent -NH2 group of glutamine may be superimposed upon the tau-N. This suggests that it is the pi-N of histidine that hydrogen bonds with ATP and that there is no significant electrostatic interaction between the histidine and ATP. Incorporating the Pro-51 mutation into each of the Asn-48 and Gln-48 mutants gives an improvement in the affinity of the enzyme for ATP, but this improvement is less than that seen with the wild-type enzyme.