Abstract
Abstract The effect of catalytic and allosteric site ligands upon the transient kinetics of formation of the carboxyenzyme complex of l-tryptophan-2,3-dioxygenase (EC 1.13.1.12) from Pseudomonas acidovorans has been studied by laser pulse, flash photolysis. Previous studies demonstrated that saturation of the catalytic and allosteric sites of the enzyme by l-tryptophan resulted in a 28-fold increase in the equilibrium constant for carboxyenzyme complex formation (Maeno, H., and Feigelson, P. (1968) J. Biol. Chem. 243, 301–305). The present studies have shown that the predominant change brought about by l-tryptophan is an 18- to 20-fold increase in in the rate constant for the reaction, while the rate constant for the off reaction decreases only slightly. With the use of the nonsubstrate analogues of l-tryptophan, 5-fluorotryptophan (specific ligand for the catalytic site) and α-methyltryptophan (ligand for the allosteric site), it was possible to determine separately the effects on kon and koff of saturation of the two sites. Saturation of the allosteric site alone with α-methyltryptophan does not affect Keq or kon, i.e. does not cause conversion of tryptophan oxygenase from the slow reacting species to the rapid reacting species. Saturation of the catalytic site alone with 5-fluorotryptophan does increase Keq and kon. At a nonsaturating level of 5-fluorotryptophan saturation of the allosteric site with α-methyltryptophan serves to increase the proportion of the enzyme present as the rapid reacting species. Thus, the consequences of the binding of l-tryptophan or α-methyltryptophan to the allosteric site of tryptophan oxygenase is to alter the enzyme increasing the affinity of its catalytic site for l-tryptophan or 5-fluorotryptophan, which in turn increases the proportion of the enzyme which reacts rapidly with CO.
Highlights
Has been studied by laser pulse, flash photolysis
The equilibrium constant for this carboxyenzyme complex formation has been determined for the pseudomonad enzyme [3], and the consequences of saturation of the catalytic site is a 2%fold augmentation in the affinity of the ferroheme enzyme for CO
As L-tryptophan binds to both the allostcric and catalytic sites of the enzyme, comparable studies eml)loying tryptophan analogues which respectively bind exclusively to the catalytic site, 5-fluorotryptophan [4], and to the allosteric site, cu-methyltrgptophan (II), should elucidate the conformational and kinetic consequences of the respective saturation of these sites
Summary
The equilibrium constant for this carboxyenzyme complex formation has been determined for the pseudomonad enzyme [3], and the consequences of saturation of the catalytic site is a 2%fold augmentation in the affinity of the ferroheme enzyme for CO [3, 4]. Since the equilibrium constant is known, the rate constant for the “off” reaction could be calculated directly In this way, the effects of saturation of the catalytic or allosteric, or both, sites of tryptophan oxygenase on the rate constant for the formation of the carboxyfcrroheme enzyme complex could be directly observed and the effects on the dissociation of the carboxyenzyme complex calculated
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