Dihydro-orotase from Clostridium oroticum. Purification and reversible removal of essential zinc.
A new purification procedure involving five column-chromatography steps is described for dihydro-orotase (L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3) from Clostridium oroticum (A.T.C.C. 25750). The native purified enzyme is a dimer of Mr 102 000 and contains 4.0 +/- 0.3 g-atoms of zinc/mol of dimer. These observations agree with those reported previously [Taylor, Taylor, Balch & Gilchrist (1976) J. Bacteriol. 127, 863-873]. It is conclusively demonstrated that dihydro-orotase is a zinc metalloenzyme. Zinc is reversibly removed by treatment with chelators in phosphate buffer at pH 6.5, as demonstrated by atomic absorption spectrophotometry and decrease of enzyme activity. The specific activity is linearly dependent on zinc content. Addition of ZnSO4 to the chelator-treated enzyme results in regain of the normal complement of zinc and enzyme activity. Kinetic properties of the reconstituted enzyme are indistinguishable from those of the native enzyme. The amino acid composition of the homogeneous enzyme suggests that the zinc atoms occupy different environments.
Pettigrew
D W
DW
Bidigare
R R
RR
Mehta
B J
BJ
Williams
M I
MI
Sander
E G
EG
D000581:Amidohydrolases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D002853:Chromatography, Liquid; D003013:Clostridium / Q000201:enzymology*; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D015032:Zinc / Q000032:analysis*
D000596:Amino Acids; D004492:Edetic Acid; D000581:Amidohydrolases; D004080:Dihydroorotase; D015032:Zinc