Oxidation-reduction properties of the regulatory disulfides of sorghum chloroplast nicotinamide adenine dinucleotide phosphate-malate dehydrogenase

Biochemistry. 2000 Mar 28;39(12):3344-50. doi: 10.1021/bi9916731.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alanine / genetics
  • Chloroplasts / enzymology*
  • Chloroplasts / genetics
  • Cysteine / chemistry
  • Cysteine / genetics
  • Disulfides / chemistry*
  • Disulfides / metabolism
  • Edible Grain / enzymology
  • Edible Grain / genetics
  • Enzyme Activation / genetics
  • Hydrogen-Ion Concentration
  • Malate Dehydrogenase (NADP+)
  • Malate Dehydrogenase / chemistry*
  • Malate Dehydrogenase / genetics
  • Malate Dehydrogenase / metabolism
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Titrimetry

Substances

  • Disulfides
  • Recombinant Proteins
  • Malate Dehydrogenase
  • Malate Dehydrogenase (NADP+)
  • Cysteine
  • Alanine