Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum.
Enzyme elements that are involved in the reversible cyclization of L-carbamylaspartate to L-dihdroorotate catalyzed by dihydroorotase (EC 3.5.2.3) from Clostridium oroticum (ATCC 25750) have been studied. Removal of Zn(II) from the enzyme by chelators followed by incubation of apoenzyme with Co(II) results in replacement of two to three of the four Zn(II) ions per molecule by Co(II). The catalytic properties of the Zn(II)Co(II) dihydroorotase are different from those of native enzyme. The Vmax is increased for both the synthesis and hydrolysis of L-dihydroorotate. The Km for L-dihydroorotate is unchanged, while the Km for L-carbamylaspartate is increased more than twofold. On the other hand, the kinetic properties of Zn(II)-reconstituted dihydroorotase are indistinguishable from those of native enzyme. The pH dependence of Vmax is also altered by the Co(II) substitution. For both Zn(II)- and Zn(II)Co(II)-dihydroorotase, this pH dependence is well described by a single ionization and the pK's for L-dihydroorotate synthesis and hydrolysis are different. Substitution with Co(II) increases the pK for both reaction directions to different extents. These results strongly support a role for the tightly bound metals in the catalytic mechanism. In addition, diethylpyrocarbonate rapidly inactivates the enzyme. The inactivation is prevented by L-dihydroorotate. This result is consistent with a role for at least one histidine in catalysis. The possibility that C. oroticum dihydroorotase may be useful model for the more complex mammalian enzyme is considered.
Pettigrew
D W
DW
Mehta
B J
BJ
Bidigare
R R
RR
Choudhury
R R
RR
Scheffler
J E
JE
Sander
E G
EG
D000581:Amidohydrolases / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D003035:Cobalt; D003500:Cyclization; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006639:Histidine / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D015032:Zinc / Q000502:physiology
C004768:4,5-dihydroorotic acid; D001224:Aspartic Acid; D003035:Cobalt; D006639:Histidine; D009963:Orotic Acid; D000581:Amidohydrolases; D004080:Dihydroorotase; D015032:Zinc; D004047:Diethyl Pyrocarbonate; C006893:ureidosuccinic acid