Serine319 and 321 Are Functional in Isocitrate Lyase from Escherichia coli (original) (raw)
With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichia coli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions. Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine appreciably reduced the k cat of E. coli isocitrate lyase relative to that for wild-type (100) as follows: