Transition State Stabilization by the N-terminal
Anticodon-binding Domain of Lysyl-tRNA Synthetase*,
Teisuke
Takita
and
Kuniyo
Inouye
From the Division of Food Science and Biotechnology,
Graduate School of Agriculture, Kyoto University, Kitashirakawa,
Kyoto 606-8502, Japan
Lysyl-tRNA synthetase from Bacillus
stearothermophilus (B.s. LysRS) (EC 6.1.1.6)
catalyzes aminoacylation of tRNALys with
L-lysine, in which L-lysine was first activated
with ATP to yield an enzyme (lysyladenylate complex), and then the
lysine molecule was transferred from the complex to
tRNALys. B.s. LysRS is a homodimeric enzyme
with a subunit that consists of two domains, an N-terminal tRNA
anticodon-binding domain (TAB-ND: Ser1-Pro144)
and a C-terminal Class II-specific catalytic domain (CAT-CD: Lys151-Lys493). CAT-CD alone retained catalytic
activity, although at a low level; TAB-ND alone showed no activity.
Size exclusion chromatography revealed that CAT-CD exists as a dimer,
whereas TAB-ND was a monomer. The formation of a complex consisting of
these domains was detected with the guidance of surface plasmon
resonance. In accordance with this, the addition of TAB-ND to CAT-CD
significantly enhanced both the L-lysine activation and the
tRNA aminoacylation reactions. Kinetic analysis showed that deletion of
TAB-ND resulted in a significant destabilization of the transition
state of CAT-CD in the L-lysine activation reaction but had
little effect on the ground state of substrate binding. A significant
role of a cross-subunit interaction in the enzyme between TAB-ND and
CAT-CD was proposed for the stabilization of the transition state in
the L-lysine activation reaction.
*
This work was supported in part by Grants-in-Aid 10760049 and 12760055 for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to T. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains text, equations, and Figs. 1-4.
To whom correspondence should be addressed. Tel.: 81-75-753-6268;
Fax: 81-75-753-6265; E-mail: takita@kais.kyoto-u.ac.jp.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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