| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 84 === Cytosolic malic enzyme [(S)-malate:NADP+ oxidoreductase
(oxaloacetate-decarboxylating), EC 1.1.1.40] decarboxylates L-
malate to yield pyruvate and CO2 with concomitant reduction of
NADP+ to NADPH. This generated NADPH is a source of the
reducing equivalents necessary for the de novo biosynthesis
of long-chain fatty acid. The pigeon liver cytosolic malic
enzyme is a tetramer composed of four identical subunits with
molecular weight about 65,000 dalton. Based on the chemical
modification studies, histidine and arginine residues were
proposed to involve in the NADP+ binding and L-malate,
respectively. Aligning of amino acid sequence of pigeon liver
cytosolic malic enzyme with those from other species,
twenty-two arginine and six histidine residues were located
in the highly conserved regions. These highly conserved amino
acid residues were mutated by the alanine scanning
mutagenesis. These mutants were the subjects for preliminary
kinetic studies. The apparent Km values for L-malate for R24A
and R70A is increased by about 5-fold and 10-fold,
respectively. Both the apparent Km values for Mn2+ for R24A
and R70A are increased by about 10-fold. The kcat values for
R24A and R70A are decreased by 10-fold and 1.5-fold,
respectively. The kinetic parameters of all other mutants are
similar to those for wild type. These results indicate that
both of R24 and R70 may be involve in L-malate binding. The
KmNADP+ values of all the mutants are almost identical to
that of the wild type. It suggested that these amino acid
residues may not involve in the nucleotide binding. Not all
mutants showed as tetramer on native PAGE gel. All the
mutants showed one protein band in molecular weight about 65
,000 dalton on SDS PAGE gel. These results appear to indicat
that these amino acid residues may participate in subunit''s
interaction.
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