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The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli

The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli

Archives of Biochemistry and Biophysics 336(2): 309-315

Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of k-cat compared to those for wild-type (wt) enzyme (100) at 37 degree C, pH 7.3, are 18, 1, lt 1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. K-m values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log k-cat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pK-a and pK-b values for dissociation of an ionizable group on the enzyme substrate complex of lt 6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pK-m vs pH were different for the wt and H356K variant. Values of pK-a and pK-b (derived from log k-cat/K-m plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pK-b value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.

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Accession: 010002015

Download citation: RISBibTeXText

PMID: 8954579

DOI: 10.1006/abbi.1996.0562

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