2ND-SITE SUPPRESSION OF REGULATORY PHOSPHORYLATION IN ESCHERICHIA-COLI ISOCITRATE DEHYDROGENASE

Inactivation of Escherichia coli isocitrate dehydrogenase upon phospho rylation at S113 depends upon the direct electrostatic repulsion of th e negatively charged gamma-carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (k(cat)/Ki . isocitra t/Km . NADP) by a factor of 10(7). Here, we demonstrate that the inact ivating effects of the electrostatic repulsion are completely eliminat ed by a second-site mutation, and provide the structural basis for thi s striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D-helix, interacts with isocitrate and NADP(+), an d has been postulated to serve in both substrate binding and in cataly sis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effe cts of S113D or S113E: the performance of the double mutants is 10(5) higher than the S113D and S113E single mutants. These mutations have l ittle effect on the kinetics of alternative substrates, which lack the charged gamma-carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the c rystal structure of the N115L mutant, the leucine adopts a different c onformer from the wild-type asparagine. Repacking around the leucine f orces the amino-terminus of the D-helix away from the rest of the acti ve site. The hydrogen bond between E113 and N115 in the S113E single m utant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the gamma-carboxylate of isocitrate. These mo vements increase the distance between the carboxylates, diminish the e lectrostatic repulsion, and lead to the remarkably high activity of th e S113E/N115L mutant.