Kinetic mechanism of glutathione conjugation to leukotriene A4 by leukotriene C4 synthase.
The kinetic mechanism for human leukotriene (LT) C4 synthase, a membrane-bound glutathione S-transferase, which catalyzes the conjugation of glutathione (GSH) to 5,6-oxido-7,9,11, 14-eicosatetraenoic acid (LTA4), to form 5(S)-hydroxy-6(R)-S-glutathionyl-7,9,trans-11, 14-cis-eicosatetraenoic acid (LTC4) was investigated by initial rate kinetic studies in which concentrations of both substrates and the reversible dead-end inhibitor, 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)- methoxy]- 4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]ethoxy]butanoic acid (L-699,333) were varied. Analysis of the initial velocities of LTC4 formation in the absence of the inhibitor using non-linear regression fits of various models to the data favoured a random, rapid equilibrium mechanism, with strong substrate inhibition by LTA4, over both a compulsory ordered mechanism and a ping-pong mechanism. The estimated parameters were calculated to be Vmax = 14 +/- 4 microM/min, KLTA4 = 40 +/- 18 microM, KGSH = 0.4 +/- 0.2 mM, and a KiLTA4 = 2.3 +/- 1.7 microM for the rapid equilibrium random model. Inhibition of enzymatic activity by L-699,333 was found to be reversible as assessed by the ability of the enzyme to restore its activity by 95% upon dilution. L-699,333 was found to be a competitive inhibitor against GSH and non-competitive against LTA4. Non-linear least squares regression analysis yielded estimated parameters of Km = 0.7 +/- 0.1 mM, Vmax = 2.5 +/- 0.1 microM/min, and Ki = 0.7 +/- 0.1 microM for GSH at a fixed LTA4 concentration of 20 microM, and Km = 45 +/- 3 microM, Vmax = 4.9 +/- 0.2 microM/min, and a Ki = 5.8+/-0.4 microM for LTA4 at a fixed GSH concentration of 2 mM. The rate equation for the random equilibrium mechanism accommodates the inhibition patterns observed for L-699,333 against both substrates as revealed by kinetic fits of the inhibition data to the overall rate equation.
Gupta
N
N
Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond, Montreal, Quebec, Canada. namrata_gupta@merck.com
Gresser
M J
MJ
Ford-Hutchinson
A W
AW
D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D017572:Leukotriene A4 / Q000378:metabolism*; D008954:Models, Biological; D011725:Pyridines / Q000494:pharmacology; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D007211:Indoles; D017572:Leukotriene A4; D011725:Pyridines; C086903:L 699333; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D005978:Glutathione