Allosteric modulation of the transient kinetics of carboxytryptophan oxygenase complex formation after laser flash photolysis.

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 1% to 20-fold increase in in the rate constant for the “on” reaction, while the rate constant for the “off” reaction decreases only slightly. With the use of the nonsubstrate analogues of L-tryptophan, S-fluorotryptophan (specific ligand for the catalytic site) and cr-methyltryptophan (ligand for the allosteric site), it was possible to determine separately the effects on k,, and koff of saturation of the two sites. Saturation of the allosteric site alone with o+methyltryptophan does not affect Keq or k,,, 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 with5fluorotryptophan does increase Kc4 and k,,. At a nonsaturating level of 5-fluorotryptophan saturation of the allosteric site with or-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 a-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.