The Consequences of Replacing Histidine 356 in Isocitrate Lyase fromEscherichia coli

Isocitrate lyase fromEscherichia colihas been expressed in transformedE. coliJE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis oficlby 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 ofkcatcompared to those for wild-type (wt) enzyme (100) at 37°C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively.Kmvalues 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 logkcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKaand pKbvalues for dissociation of an ionizable group on the enzyme–substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKmvs pH were different for the wt and H356K variant. Values of pKaand pKb(derived from logkcat/Kmplots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKbvalue 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.