Histidyl transfer ribonucleic acid synthetase from Salmonella typhimurium. Interaction with substrates and ATP analogs

Structural requirements for substrate binding to histidyl-tRNA synthetase from S. typhimurium were investigated using ATP analogs. Ki values and the relative binding affinity of the enzyme for these analogs were detd. in the tRNA aminoacylation reaction. The enzyme was highly specific for ATP: no binding was found for GTP, CTP, TTP, and UTP. DATP was a very poor substrate for acylation of tRNA, with a Km 40-fold higher than that of ATP. Binding of ATP requires interactions of the NH2 group of adenosine and the sugar moiety; the 2' and the 5' positions of the ribose appear to be essential for recognition; the phosphate groups enhance the binding. AMP was a noncompetitive inhibitor with ATP. The interaction of histidyl-tRNA synthetase, a dimeric enzyme, with histidine and ATP was examd. by fluorescence measurements at equil. and by equil. dialysis. Binding with L-histidine was significantly tighter at pH 6 than at pH 7, while the ATP binding was independent of pH. The stoichiometry was measured at pH 7.5 by equil. dialysis and was 1 mole ATP/mole enzyme and, variably, close to 2 or 1 mole histidine/mole enzyme. [on SciFinder(R)]