Rat liver gamma-butyrobetaine hydroxylase catalyzed reaction: influence of potassium, substrates, and substrate analogues on hydroxylation and decarboxylation.
Interaction of rat liver gamma-butyrobetaine hydroxylase (EC 1.14.11.1) with various ligands was studied by following the decarboxylation of alpha-ketoglutarate, formation of L-carnitine, or both. Potassium ion stimulates rat liver gamma-butyrobetaine hydroxylase catalyzed L-carnitine synthesis and alpha-ketoglutarate decarboxylation by 630% and 240%, respectively, and optimizes the coupling efficiency of these two activities. Affinities for alpha-ketoglutarate and gamma-butyrobetaine are increased in the presence of potassium. gamma-Butyrobetaine hydroxylase catalyzed decarboxylation of alpha-ketoglutarate was dependent on the presence of gamma-butyrobetaine, L-carnitine, or D-carnitine in the reaction and exhibited Km(app) values of 29, 52, and 470 microM, respectively. gamma-Butyrobetaine saturation of the enzyme indicated a substrate inhibition pattern in both the assays. Omission of potassium decreased the apparent maximum velocity of decarboxylation supported by all three compounds by a similar percent. beta-Bromo-alpha-ketoglutarate supported gamma-butyrobetaine hydroxylation, although less effectively than alpha-ketoglutarate. The rat liver enzyme was rapidly inactivated by 1 mM beta-bromo-alpha-ketoglutarate at pH 7.0. This inactivation reaction did not show a rate saturation with increasing concentrations of beta-bromo-alpha-ketoglutarate. None of the substrates or cofactors, including alpha-ketoglutarate, protected the enzyme against this inactivation. Unlike beta-bromo-alpha-ketoglutarate, beta-mercapto-alpha-ketoglutarate did not replace alpha-ketoglutarate as a cosubstrate. Both beta-mercapto-alpha-ketoglutarate and beta-glutathione-alpha-ketoglutarate were noncompetitive inhibitors with respect to alpha-ketoglutarate.
Wehbie
R S
RS
Institute for Enzyme Research, University of Wisconsin, Madison 53705.
Punekar
N S
NS
Lardy
H A
HA
D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives; D003653:Decarboxylation; D006900:Hydroxylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D051381:Rats; D013379:Substrate Specificity; D050563:gamma-Butyrobetaine Dioxygenase
D001622:Betaine; D006899:Mixed Function Oxygenases; D050563:gamma-Butyrobetaine Dioxygenase; D011188:Potassium