Isolation and biochemical characterization of maleic-acid hydratase, an iron-requiring hydro-lyase.
A procedure for the isolation of maleic acid hydratase (D-malate hydro-lyase, EC 4.2.1.31) of about 95% purity from rabbit kidneys is described. The enzyme consists of a single polypeptide chain of 582 amino-acid residues with an approximate molecular mass of 68 kDa. The enzyme is very unstable and has an absolute requirement for chloride ions. Addition of sodium sulphide during the purification process was essential to maintain the enzyme in an activatable state. The pure preparation has low activity but responds to activation with Fe2+ ions, Na2S and a thiol. The sequence of adding the activating reagents is critical to achieve optimal activity. Ni2+ and to a lesser extent Co2+ can replace iron in the activation process. The enzyme incorporates 4-5 mol iron/mol and 4.5-6 mol sulphide/mol during activation. In this process an [Fe-S] cluster appears to be built up, as indicated by optical and electron paramagnetic resonance (EPR) spectroscopy. In activated samples exposed to air the [Fe-S] cluster is EPR-detectable through an axial signal with g = 2.01 and g = 2.029 whose temperature and power saturation characteristics were similar to those of other [3Fe-xS] clusters. The activated enzyme, however, is readily inactivated even upon minor manipulation with destruction of the iron-sulfur core.
Dreyer
J L
JL
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000838:Anions / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006836:Hydro-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins; D007668:Kidney / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000302:isolation & purification; D008293:Malates / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D010100:Oxygen / Q000502:physiology; D011817:Rabbits; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D000596:Amino Acids; D000838:Anions; D002614:Chelating Agents; D007506:Iron-Sulfur Proteins; D008293:Malates; D008670:Metals; D013439:Sulfhydryl Reagents; C030298:malic acid; D007501:Iron; D008291:Malate Dehydrogenase; D006836:Hydro-Lyases; C021529:maleate hydratase; D010100:Oxygen