Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.
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.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D008239:Lysine / Q000032:analysis*; D008291:Malate Dehydrogenase / Q000032:analysis*; D008931:Mitochondria, Muscle / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D009243:NAD; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011732:Pyridoxal Phosphate; D013552:Swine; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D009243:NAD; D011732:Pyridoxal Phosphate; D008291:Malate Dehydrogenase; D014357:Trypsin; D008239:Lysine