Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.
Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism*; D008027:Light; D010649:Phenylalanine / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism* / Q000528:radiation effects; D012343:RNA, Transfer / Q000378:metabolism; D012395:Rose Bengal; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D012395:Rose Bengal; D010649:Phenylalanine; D006639:Histidine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase; D004047:Diethyl Pyrocarbonate