JBC, Vol. 250, Issue 17, 6659-6665, Sep, 1975
Structure-activity relationships in tryptophanyl transfer ribonucleic acid synthetase from beef pancreas. Influence of the alkylation of the sulfhydryl groups on the dimer-monomer equilibrium
F. Iborra, B. Labouesse and J. Labouesse
Upon reaction with N-ethylmaleimide, tryptophanyl-tRNA synthetase from beef
pancreas dissociates into subunits. At pH7, the rate of the dissociation is
close to both the reaction rate of the buried--SH groups and the rate of
inactivation (Iborra, F., Mourgeon, G., Labouesse B., and Labouesse, J.
(1973) Eur. J. Biochem. 39, 547-556). The pH and enzyme concnetration
dependences of the reaction rate of the 16 cysteinyl residues of the enzyme
as well as that of its inactivation support the idea that inactivation by
alkylation of the--SH groups is due essentially to the dissociation of the
protein into inactive subunits and not to the chemical blocking of a
catalytic residue. This is confirmed by the independence on
N-ethylmaleimide concentration of the reaction of the buried--SH groups and
of the inactivation of the enzyme at high N-ethylmaleimide concentration.
The dissociation becomes in this case the rate-limiting step of the
chemical reaction. The monomeric structure is stabilized by the blocking of
the--SH groups exposed during the dissociation. The dissociation constant
of the dimeric enzyme is progressively increased during the alkylation. The
tightness of the associated structure depends on the protonation of groups
titrating between pH 7 and pH 9.
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