Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.
نویسندگان
چکیده
The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.
منابع مشابه
The three-dimensional structure of porcine heart mitochondrial malate dehydrogenase at 3.0-A resolution.
In a previous study, we reported the apparent similarity between a low resolution electron density map of mitochondrial malate dehydrogenase and a model of cytoplasmic malate dehydrogenase (Roderick, S. L., and Banaszak, L. J. (1983) J. Biol. Chem. 258, 11636-11642). We have since determined the polypeptide chain conformation and coenzyme binding site of crystalline porcine heart mitochondrial ...
متن کاملReversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate.
1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial ...
متن کاملRefined crystal structure of mitochondrial malate dehydrogenase from porcine heart and the consensus structure for dicarboxylic acid oxidoreductases.
The crystal structure of mitochondrial malate dehydrogenase from porcine heart contains four identical subunits in the asymmetric unit of a monoclinic cell. Although the molecule functions as a dimer in solution, it exists as a tetramer with 222 point symmetry in the crystal. The crystallographic refinement was facilitated in the early stages by using weak symmetry restraints and molecular dyna...
متن کاملInvestigation of the anticooperative binding of NADH to porcine heart mitochondrial malate dehydrogenase.
Recent studies in this laboratory have shown that porcine heart mitochondrial malate dehydrogenase (Lmalate:NAD* oxidoreductase, EC 1.1.1.37) exhibits both a concentration and a pH-dependent dissociation (Bleile, D. M., Schulz, R. A., Gregory, E. M., and Harrison, J. H. (1977) J. Biol. Chem. 252,756758; Hodges, C. T., Wiggins, J. C., and Harrison, J. H. (1977) J. BioZ. Chem. 252, 6038-6041). Si...
متن کاملMalate dehydrogenase: isolation from E. coli and comparison with the eukaryotic mitochondrial and cytoplasmic forms.
Escherichia coli malate dehydrogenase has been isolated in homogeneous form by a procedure employing chromatography on DEAE-cellulose, 5-'AMP-Sepharose, and Sephacryl-200. It is composed of two identical polypeptide chains each of Mr = 32 500. Like porcine mitochondrial malate dehydrogenase, it is devoid of tryptophan, but otherwise it is not particularly more similar in composition to one of t...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- The Journal of biological chemistry
دوره 250 22 شماره
صفحات -
تاریخ انتشار 1975