Influence of Magnesium on the Steady‐State‐Derived Order of Substrate Addition and Product Release in tRNA ^Trp Aminoacylation by Beef Pancreas Tryptophan: tRNA Ligase

Abstract

The order of the partial reactions in the aminoacylation of tRNA Trp beef by tryptophan : tRNA ligase from beef pancreas has been explored by measurements of intial velocity and product inhibition according to the analytical framework described by Cleland [ Biochim. Biophys. Acta. 67 , 104– 137, 173– 187, 188– 196 (1963)]. The kinetic patterns are suggestive of a ping‐pong mechanism (bi‐uni uni‐bi). The oreder of addition of tryptophan and ATP that one can derive is crucially dependent on the conditions of magnesium concentration used. Random fixation of tryptophan and ATP is observed in studies performed at catalytically optimal magnesium concentration. Linear‐ordered fixation of the two substrates, ATP binding first, is observed under conditions where magnesium concentration is in excess, which are not catalytically optimal. The analysis of the inhibition patterns taking into account the calculated concentrations of teh main active species Mg · ATP 2− and Mg · P 2 O 2− 7 favors the random substrate‐binding mechanism derived under optimal magnesium conditions. Under both conditions, adjusted and fixed magnesium, the release of products, AMP and tryptophanyl‐tRNA Trp beef is, ordered, AMP released first. The change in the product nhibition patterns of aminoacylation of tRNA Trp beef by beef pancreas tryptophan: tRNA ligase under different magnesium concentration conditions casts some doubt on the significance of the comparison of mechanisms derived for amino‐acid: tRNA ligases of different sources.