THE IMPORTANCE OF 4 HISTIDINE-RESIDUES IN ISOCITRATE LYASE FROM ESCHERICHIA-COLI

By site-directed mutagenesis, substitutions were made for His-184 (H-1 84), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced e nzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an i nactive isocitrate lyase, and mutation of H-184 to Gin resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electroph oresis demonstrated that isocitrate lyase containing the Lys, Arg, Gin , and Leu substitutions at H-184 was assembled poorly into the tetrame ric subunit complex. Mutation of H-197 to Lys, Arg, Leu, and Gin resul ted in an assembled enzyme with less than 0.25% wild-type activity. Fi ve substitutions for H-266 (Asp, Glu, Val, Ser, and Lys), four substit utions for H-306 (Asp, Glu, Val, and Ser), and a variant in which both H-266 and H-306 were substituted for showed little or no effect on en zyme activity. All the H-197, H-266, and H-306 mutants supported the g rowth of isocitrate lyase-deficient E. coli JE10 on acetate as the sol e carbon source; however, the H-184 mutants did not.