KINETIC MECHANISM OF GLUTATHIONE CONJUGATION TO LEUKOTRIENE A(4) BY LEUKOTRIENE C-4 SYNTHASE

The kinetic mechanism for human leukotriene (LT) C-4 synthase, a membr ane-bound glutathione S-transferase, which catalyzes the conjugation o f glutathione (GSH) to 5,6-oxide-7,9,11,14-eicosatetraenoic acid (LTA( 4)), to form 5(S)-hydroxy-6( -glutathionyl-7,9-trans-11,14-cis-eicosat etraenoic acid (LTC4) was investigated by initial rate kinetic studies in which concentrations of both substrates and the reversible dead-en d inhibitor, -2-yl)-methoxy]-4,5-dihydro-1H-thiopyrano[2,3,4-c, d]indo l-2-yl]ethoxy]butanoic acid (L-699,333) were varied. Analysis of the i nitial velocities of LTC4 formation in the absence of the inhibitor us ing non-linear regression fits of various models to the data favoured a random. rapid equilibrium mechanism, with strong substrate inhibitio n by LTA(4), over both a compulsory ordered mechanism and a ping-pong mechanism. The estimated parameters were calculated to be V-max = 14 /- 4 mu M/min, K-LTA4 = 40 +/- 18 mu M, K-GSH = 0.4 +/- 0.2 mM, and a K-iLTA4 = 2.3 +/- 1.7 eta M for the rapid equilibrium random model. In hibition 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 9 5% upon dilution. L-699,333 was found to be a competitive inhibitor ag ainst GSH and non-competitive against LTA(4). Non-linear least squares regression analysis yielded estimated parameters of K-m = 0.7 +/- 0.1 mM, V-max = 2.5 +/- 0.1 mu M/min, and K-i = 0.7 +/- 0.1 mu M for GSH at a fixed LTA(4) concentration of 20 mu M, and K-m = 45 +/- 3 mu M, V -max = 4.9 +/- 0.2 mu M/min, and a K-i = 5.8 +/- 0.4 mu M for LTA(4) a t a fixed GSH concentration of 2 mM. The rate equation for the random equilibrium mechanism accommodates the inhibition patterns observed fo r L-699,333 against both substrates as revealed by kinetic fits of the inhibition data to the overall rate equation. (C) 1998 Elsevier Scien ce B.V.