A second dihydroorotate dehydrogenase (Type A) of the human pathogen Enterococcus faecalis: expression, purification, and steady-state kinetic mechanism.
Arch Biochem Biophys
; 377(1): 178-86, 2000 May 01.
Article
in En
| MEDLINE
| ID: mdl-10775458
ABSTRACT
We report the identification, expression, and characterization of a second Dihydroorotate dehydrogenase (DHODase A) from the human pathogen Enterococcus faecalis. The enzyme consists of a polypeptide chain of 322 amino acids that shares 68% identity with the cognate type A enzyme from the bacterium Lactococcus lactis. E. faecalis DHODase A catalyzed the oxidation of l-dihydroorotate while reducing a number of substrates, including fumarate, coenzyme Q(0), and menadione. The steady-state kinetic mechanism has been determined with menadione as an oxidizing substrate at pH 7.5. Initial velocity and product inhibition data suggest that the enzyme follows a two-site nonclassical ping-pong kinetic mechanism. The absorbance of the active site FMN cofactor is quenched in a concentration-dependent manner by titration with orotate and barbituric acid, two competitive inhibitors with respect to dihydroorotate. In contrast, titration of the enzyme with menadione had no effect on FMN absorbance, consistent with nonoverlapping binding sites for dihyroorotate and menadione, as suggested from the kinetic mechanism. The reductive half-reaction has been shown to be only partially rate limiting, and an attempt to evaluate the slow step in the overall reaction has been made by simulating orotate production under steady-state conditions. Our data indicate that the oxidative half-reaction is a rate-limiting segment, while orotate, most likely, retains significant affinity for the reduced enzyme, as suggested by the product inhibition pattern.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Oxidoreductases
/
Enterococcus faecalis
/
Oxidoreductases Acting on CH-CH Group Donors
Language:
En
Journal:
Arch Biochem Biophys
Year:
2000
Document type:
Article
Affiliation country:
United States