Site-directed mutagenesis of lysine 193 in Escherichia coli isocitrate lyase by use of unique restriction enzyme site elimination.
By a newly developed double-stranded mutagenesis technique, histidine (H), glutamate (E), arginine (R) and leucine (L) have been substituted for the lysyl 193 residue (K-193) in isocitrate lyase from Escherichia coli. The substitutions for this residue, which is present in a highly conserved, cationic region, significantly affect both the Km for Ds-isocitrate and the apparent kcat of isocitrate lyase. Specifically, the conservative substitutions, K-193-->H (K193H) and K193R, reduce catalytic activity by ca. 50- and 14-fold, respectively, and the nonconservative changes, K193E and K193L, result in assembled tetrameric protein that is completely inactive. The K193H and K193R mutations also increase the Km of the enzyme by five- and twofold, respectively. These results indicate that the cationic and/or acid-base character of K193 is essential for isocitrate lyase activity. In addition to the noted effects on enzyme activity, the effects of the mutations on growth of JE10, an E. coli strain which does not express isocitrate lyase, were observed. Active isocitrate lyase is necessary for E. coli to grow on acetate as the sole carbon source. It was found that a mutation affecting the activity of isocitrate lyase similarly affects the growth of E. coli JE10 on acetate when the mutated plasmid is expressed in this organism. Specifically, the lag time before growth increases over sevenfold and almost twofold for E. coli JE10 expressing the K193H and K193R isocitrate lyase variants, respectively. In addition, the rate of growth decreases by almost 40-fold for E. coli JE10 cells expressing form K193H and ca. 2-fold for those expressing the K193R variants. Thus, the onset and rate of E. coli growth on acetate appears to depend on isocitrate lyase activity.
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
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P
Diehl
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McFadden
D000595:Amino Acid Sequence; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007522:Isocitrate Lyase / Q000737:chemistry / Q000235:genetics*; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D004262:DNA Restriction Enzymes; D007522:Isocitrate Lyase; D008239:Lysine