Oxidation-reduction properties of the regulatory disulfides of sorghum chloroplast nicotinamide adenine dinucleotide phosphate-malate dehydrogenase.
Oxidation-reduction midpoint potentials (E(m)) have been measured for the thioredoxin-dependent, reductive activation of sorghum nicotinamide adenine dinucleotide phosphate- (NADP-) dependent malate dehydrogenase (MDH) in the wild-type enzyme and in a number of site-specific mutants. The E(m) value associated with activation of the wild-type enzyme, -330 mV at pH 7.0, can be attributed to the E(m) of the C365/C377 disulfide present in the C-terminal region of the enzyme. The C24/C29 disulfide, located in the N-terminal region of the enzyme and the only other disulfide present in oxidized, wild-type MDH, has a E(m) value of -280 mV at pH 7.0. A third regulatory disulfide, C24/C207, that is absent in the oxidized enzyme but is thought to be formed during the activation process, has an E(m) value at pH 7.0 of -310 mV. E(m) vs pH profiles suggest pK(a) values for the more acidic cysteine involved in the formation of each of these disulfides of 8.5 for C24/C29; 8.1 for C24/C207; and 8.7 for C365/C377. The results of this study show that the N-terminal disulfide formed between C24 and C29 has a more positive E(m) value than the two other disulfides and is thus is likely to be the "preregulatory disulfide" postulated to function in activating the enzyme.
Hirasawa
M
M
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
Ruelland
E
E
Schepens
I
I
Issakidis-Bourguet
E
E
Miginiac-Maslow
M
M
Knaff
D B
DB
D000409:Alanine / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D002523:Edible Grain / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+); D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017186:Titrimetry
D004220:Disulfides; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D003545:Cysteine; D000409:Alanine