Aldolase A Ins(1,4,5)P3-binding domains as determined by site-directed mutagenesis
Aldolase A Ins(1,4,5)P3-binding domains as determined by site-directed mutagenesis
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DOI：
10.1042/0264-6021:3410805
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发表时间：
1999-08-01
影响因子：
4.1
通讯作者：
Coburn, RF
中科院分区：
文献类型：
--
作者：
Baron, CB;Tolan, DR;Coburn, RF
We substituted neutral amino acids for some positively charged residues (R42, K107, K146, R148 and K229) that line the active site of aldolase A in an effort to determine binding sites for inositol 1,4,5-trisphosphate. In addition, D33 (involved in carbon-carbon bond cleavage) was mutated. K229A and D33S aldolases showed almost no catalytic activity, but Ins(1,4,5)P-3 binding was similar to that determined with the use of wild-type aldolase A. R42A, K107A, K146R and R148A had markedly decreased affinities for Ins(1,4,5)P-3 binding, increased EC50 values for Fru(1,6)P-2-evoked release of bound Ins(1,4,5)P-3 and increased K-i values for Ins(1,4,5)P-3-evoked inhibition of aldolase activity. K146Q (positive charge removal) had essentially no catalytic activity and could not bind Ins(1,4,5)P-3. Computer-simulated docking of Ins(1,4,5)P-3 in the aldolase A structure was consistent with electrostatic binding of Ins(1,4,5)P-3 to K107, K146, R148, R42, R303 and backbone nitrogens, as has been reported for Fru(1,6)P-2 binding. Results indicate that Ins(1,4,5)P-3 binding occurs at the active site and is not dependent on having a catalytically active enzyme; they also suggest that there is competition between Ins(1,4,5)P-3 and Fru(1,6)P-2 for binding. Although Ins(1,4,5)P-3 binding to aldolase involved electrostatic interactions, the aldolase A Ins(1,4,5)P-3-binding domain did not show other similarities to pleckstrin homology domains or phosphotyrosine-binding domains known to bind Ins(1,4,5)P-3 in other proteins.
