Serine activation is the rate limiting step of tRNA^Seraminoacylation by yeast seryl tRNA synthetase

Abstract

Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.

The mechanism of salt‐induced stimulation of tRNASer aminoacylation by yeast seryl‐tRNA synthetase

“…The rate is stimulated in the presence of Blank values as measured in the absence of serine were subtraced. Evaluation of the curves yielded the initial rate of serine activation in the absence of tRNA Ser and tlre steady state rate of ATP hydrolysis in the presence of tRNA Set which is practically equal to the rate of serine activation under this condition [13].…”

“…On the basis of relaxation kinetic studies on tRNA Set dissociation from SRS [ 11 ] the stimulating effect of KCl was explained by a salt4nduced acceleration of seryl-tRNA Set dissociation [12]. However, recent investigations [13] revealed that SRS differs from other aminoacyl-tRNA synthetases in that the activation of serine is the rate limiting step of tRNA Set aminoacylation and not the dissociation of the seryl-tRNASer-sRS complex. We therefore have examined the question further and found the serine activation to occur much faster in the absence than in the presence of tRNA Ser.…”

Asymmetric Amino Acid Activation by Class II Histidyl-tRNA Synthetase from Escherichia coli

“…A detailed pre-steady-state analysis of mutants of tRNA His or HisRS compromised with respect to tRNA identity suggested that, in the complete aminoacylation reaction, formation of aminoacyl adenylate in the second active site is contingent upon a productive aminoacyl transfer reaction in the first (20). These and other data led to the proposal of an alternating site model for HisRS (20) that is analogous to the "flip flop" catalysis suggested for class II PheRS (21,22) and class Ic TrpRS (14). This raises the possibility that the catalytic cycles of dimeric class II enzymes and dimeric class Ic enzymes share some common feature.…”

Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS, from Myxococcus xanthus

“…It has been reported that inorganic pyrophosphate inhibits Ap 4 A synthesis by aminoacyl-tRNA synthetases through competitive inhibition of the ATP reaction with aminoacyl-adenylate [8,21]. To confirm whether inorganic pyrophosphate inhibits the Ap 4 A synthase activity of LysS, LysS was incubated with inorganic pyrophosphate in the presence of 5 mM ATP.…”