Kinetic mechanism of dihydropyrimidine dehydrogenase from pig liver

Journal Article · Sun Aug 05 04:00:00 EDT 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6530290

Podschun, B; Cook, P F; Schnackerz, K D ^[1]

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1. Univ. of Wuerzburg (Germany, F.R.)

Data on initial velocity and isotope exchange at equilibrium suggest a nonclassical ping-pong mechanism for the dihydropyrimidine dehydrogenase from pig liver. Initial velocity patterns in the absence of inhibitors appeared parallel at low reactant concentration, with substrate inhibition by NADPH that is competitive with uracil and with substrate inhibition by uracil that is uncompetitive with NADPH. The Km values for both uracil (1 microM) and NADPH (7 microM) are low. As a result, it was difficult to determine whether the initial velocity pattern in the absence of added inhibitors was parallel. Thus, the pattern was redetermined in the presence of the dead-end inhibitor 2,6-dihydroxypyridine, which binds to both sites. This treatment effectively eliminates the inhibition by both substrates and increases their Km values, giving a strictly parallel pattern. Product and dead-end inhibition patterns are consistent with a mechanism in which NADPH reduces the enzyme at site 1 and electrons are transferred to site 2 to reduce uracil to dihydrouracil. The predicted mechanism is corroborated by exchange between ({sup 14}C) NADP and NADPH as well as ({sup 14}C)thymine and dihydrothymine in the absence of the other substrate-product pair.

• OSTI ID: 6530290
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:22; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
AZINES
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
COENZYMES
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ENZYME INHIBITORS
ENZYMES
GLANDS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPE DILUTION
ISOTOPIC EXCHANGE
KINETICS
LABELLED COMPOUNDS
LIVER
MAMMALS
NADP
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
OXIDOREDUCTASES
PYRIMIDINES
REACTION KINETICS
SUBSTRATES
SWINE
THYMINE
TRACER TECHNIQUES
URACILS
VERTEBRATES