Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.
Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.
Velasco-García
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Laboratorio de Osmorregulación, ENEP Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios s/n, Tlalnepantla, Estado de México, C.P. 54090, México.
González-Segura
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L
Muñoz-Clares
R A
RA
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001622:Betaine / Q000031:analogs & derivatives* / Q000378:metabolism*; D050728:Betaine-Aldehyde Dehydrogenase; D002384:Catalysis / Q000187:drug effects; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013816:Thermodynamics
D009243:NAD; D001622:Betaine; D009249:NADP; D000244:Adenosine Diphosphate; C026820:betaine aldehyde; D000445:Aldehyde Oxidoreductases; D050728:Betaine-Aldehyde Dehydrogenase