Isolation and properties of oxaloacetate keto-enol-tautomerases from bovine heart mitochondria.
Two highly purified proteins with quite different properties capable of oxaloacetate keto-enol-tautomerase activity (oxaloacetate keto-enol-isomerase, EC 5.3.2.2) were isolated from the bovine heart mitochondrial matrix. The first protein has an apparent molecular mass of 37 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-200 gel filtration. It is quite stable upon storage at 40 degrees C and reaches the maximal catalytic activity at pH 8.5 with a half-maximal activity at pH 7.0. The enzyme is specifically inhibited by oxalate and diethyloxaloacetate. When assayed in the enol----ketone direction at 25 degrees C (pH 9.0), the enzyme obeys a simple substrate saturation kinetics with Km and Vmax values of 45 microM and 74 units per mg of protein, respectively; the latter value corresponds to the turnover number of 2700 min-1. The second protein has an apparent molecular mass of 80 kDa as determined by SDS-gel electrophoresis and Sephacryl SF-300 gel filtration. The enzyme is rapidly inactivated at 40 degrees C and shows a sharp pH optimum of activity at pH 9.0. The enzyme can be completely protected from thermal inactivation by oxaloacetate and dithiothreitol. The kinetic parameters of the enzyme as assayed in the enol----ketone direction at 25 degrees C (pH 9.0) are: Km = 220 microM and Vmax = 20 units per mg of protein; the latter corresponds to the turnover number of 1600 min-1. The enzyme activity is specifically inhibited by maleate and pyrophosphate. About 30% of the total oxaloacetate tautomerase activity in crude mitochondrial matrix is represented by the 37 kDa enzyme and about 70% by the 80 kDa protein.
Belikova
Y O
YO
Department of Biochemistry, School of Biology, Moscow State University, U.S.S.R.
Burov
V I
VI
Vinogradov
A D
AD
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000818:Animals; D002384:Catalysis; D002417:Cattle; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008298:Maleates / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D010070:Oxalates / Q000494:pharmacology; D019815:Oxalic Acid; D010071:Oxaloacetates / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology
D000596:Amino Acids; D008298:Maleates; D010070:Oxalates; D010071:Oxaloacetates; D010710:Phosphates; C030272:maleic acid; D019815:Oxalic Acid; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C048474:oxaloacetate tautomerase; D000645:Ammonium Sulfate; D004229:Dithiothreitol