Isocitrate lyase and the glyoxylate cycle. Progress report, February 16, 1992--February 15, 1993 Page: 2 of 6
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remarkable that the amino acid sequences outside this 100-residue insertion show
considerable similarity and four regions of homology. Given the strong evolutionary
conservation of icl function (Vanni, Giachetti, Pinzauti and McFadden, Comp. Biochem.
Physiol. 95B, 431 (1990)] it is almost certain that the conserved residues identified (1) are
involved in substrate binding or catalysis. Thus the way has been opened to direct probing
of the function of ici by amino acid replacement using directed mutagenesis.
2. Directed Mutagenesis of the Icl Gene
Recently directed mutagenesis of the ici gene from E. coli has enabled the following
his replacements: his 266 -- asp, glu, val, ser, lys and his 306 -- asp, glu, val, ser. These
replacements resulted in almost no loss in enzyme activity (10-40% loss) and little change
(<30%) in Km for isocitrate. Clearly the indication that his 266 and 306, neither of which
is conserved, were essential based upon chemical modification was incorrect. This
research is being extended to replacement of his 184 and 197, both of which are completely
conserved in an active site segment of primary structure.
Previous studies supported by this grant have established that cys195 is alkylated
by brompyruvate, a substrate analog, and that the alkylated form is inactive [Ko and
McFadden, Arch. Biochem., 278, 373 (1990)]. In directed mutagenesis studies, this
residue has been replaced in the E. coli enzyme by ser leading to inactive enzyme. In this
case, the results from directed mutagenesis corroborate the inferences from affinity labeling
that cysl95, which is completely conserved in all aligned icl sequences, is essential.
In other studies, using a newly developed double stranded mutagenesis technique,
his(H), glu (E), arg (R) and leu (L) have been substituted for the lys 193 residue (K193) in
icl from E. 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 (1). Specifically, the conservative substitutions, K193H and K193R,
reduce catalytic activity by ca. 50-fold and 14-fold respectively and the non-conservative
changes, K193E and K193L, result in assembled tetrameric protein that is completely5
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McFadden, B. A. Isocitrate lyase and the glyoxylate cycle. Progress report, February 16, 1992--February 15, 1993, report, December 31, 1992; United States. (https://digital.library.unt.edu/ark:/67531/metadc1311636/m1/2/: accessed July 16, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.