Arginyl-tRNA Synthetase from Baker's Yeast. Order of Substrate Addition and Action of ATP Analogs in the Aminoacylation Reaction; Influence of Pyrophosphate on the Catalytic Mechanism

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Abstract
The order of substrate addition to arginyl-tRNA synthetase from baker''s yeast was investigated to bisubstrate kinetics, product inhibition and inhibition by 3 different inhibiting ATP analogs, the 6-N-benzyl, 8-bromo and 3''-deoxy derivatives of ATP, each acting competitively with respect to one of the substrates. The kinetic patterns are consistent with a random ter-ter mechanism, an addition of the 3 substrates and release of the products in random order. The different inhibitors are bound to different enzyme substrate complexes of the reaction sequence. Addition of inorganic pyrophosphatase changes the inhibition patterns and addition of methylenediphosphonate as pyrophosphate analog abolishes the effect of pyrophosphatase, showing that the concentration of pyrophosphate is determinant for the mechanism of catalysis.