Class II prolyl-tRNA synthetase (ProRS) from Escherichia coli contains all three of the conserved consensus motifs characteristic of class II aminoacyl-tRNA synthetases. In this study, chemical modification and site-directed mutagenesis of the single cysteine located at position 443 in motif 3 of Escherichia coli ProRS is carried out. We show that chemical modification of C443 blocks the ability of the enzyme to form the activated aminoacyl-adenylate, a prerequisite for tRNA(Pro) aminoacylation. Nearly complete protection from inactivation is achieved by preincubating the enzyme with ATP or ATP and proline, but not proline alone or tRNA(Pro). Mutagenesis of C443 to amino acids Ala, Gly, and Ser resulted in significant decreases (16-225-fold) in k(cat)/K(M)(Pro) as measured by the ATP-PP(i) exchange reaction. The Ala and Gly mutations have a relatively small effect (4-7-fold) on the overall aminoacylation reaction, while the activity of the C443S mutant in this same assay is substantially reduced (80-fold). A sequence comparison of the motif 3 region of class II synthetases shows that C443 aligns with residues that have been implicated in amino acid binding specificity. The results of our study suggest that while the thiol located at position 443 of Escherichia coli ProRS is not essential for catalysis, this residue is likely to be in a buried region that forms the prolyl-adenylate substrate binding pocket.
The assembly kinetics of microtubule protein are altered by ionic strength, temperature and Mg2+, but not by pH. High ionic strength (I0.2), low temperature (T less than 30 degrees C) and elevated Mg2+ (greater than or equal to 1.2 mM) induce a transition from biphasic to monophasic kinetics. Comparison of the activation energy obtained for the fast biphasic step at low ionic strength (I0.069) shows excellent agreement with the values obtained at high ionic strength, low temperature and elevated Mg2+. From this observation it can be implied that the tubulin-containing reactant of the fast biphasic event is also the species that elongates microtubules during monophasic assembly. Second-order rate constants for biphasic assembly are 3.82(+/- 0.72) x 10(7) M-1.s-1 and 5.19(+/- 1.25) x 10(6) M-1.s-1, and for monophasic assembly the rate constant is 2.12(+/- 0.56) x 10(7) M-1.s-1. The microtubule number concentration is constant during elongation of microtubules for biphasic and monophasic assembly.
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