[Role of the carboxylic groups in interaction of phenylalanyl-tRNA synthetase with substrates].

The effect of modification of carboxylic groups of phenylalanyl-tRNA synthetase by p-toluene sulfonate N-cyclohexyl-N'-beta-(4-methylmorpholine) ethylcarbodiimide (CMEC) on the activity of the enzyme was investigated. It was shown that modification of two moles of carboxylic groups per mole of the enzymes leads to the diminution of negative charge of the enzyme and to inactivation in ATP-[32P]PPi-exchange and aminoacylation reactions. The inactivation is completely reversed by mild alkaline hydrolysis. ATP in concentration 2 X 10(-4) M partially protects the enzyme against inactivation, protective effect being stimulated by Mg2+ and 0.4-0.7 moles of carboxylic groups per mole of the enzyme are protected against inactivation is observed although the depth of modification is increased. Other substrates do not have protective effect. Modification of the enzyme by CMEC increases Kdiss value of [14C]-Phe-tRNA enzyme complex and Km value for tRNAPhe in aminoacylation by factor of three. Vmax for all substrates in both aminoacylation and leads to 40% increase of Hill's coefficient for ATP in ATP-[32P]PPi-exchange reaction but not in aminoacylation. The carboxylic groups modified by CMEC are assumed to take part in ATP recognition and in catalysis of the ATP conversion and in catalysis of transfer of activated amino acid residues on tRNA.