Mechanistic deductions from multiple kinetic and solvent deuterium isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Escherichia coli tryptophan indole-lyase

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Abstract
Analysis of the pH dependence of the kinetic parameters and competitive inhibitor Ki values for tryptophan indole-lyase suggests two enzymic groups must be unprotonated in order to facilitate binding and catalysis of tryptophan. The V/K for tryptophan and the pKi for oxindolyl-L-alanine, a putative transition state analogue and competitive inhibitor, decrease below two pK values of 7.6 and 6.0, while the Ki for L-alanine, also a competitive inhibitor, is 3300-fold larger (20 mM) than that for oxindolyl-L-alanine and increases below a single pK of 7.6. A single pK of 7.6 is also observed in the V/K profile for the alternate substrate, S-methyl-L-cysteine. Therefore, the enzymic group with a pK of 7.6 is responsible for proton abstraction at the 2-position of tryptophan, while the enzymic group with a pK of 6.0 interacts with the indole portion of tryptophan and probably catalyzes formation of the indolenine tautomer of tryptophan (in concert with proton transfer to C-3 of indole from the group with pK 7.6) to facilitate carbon-carbon bond cleavage and elimination of indole. The pH variation of the primary deuterium isotope effects for proton abstraction at the 2-position of tryptophan (DV=2.5 and D(V/Ktrp) = 2.8) are pH independent, while the Vmax for tryptophan or S-methyl-L-cysteine is the same and also pH independent. Thus, substrates bind only to the correctly protonated form of the enzyme. Further, tryptophan is not sticky, and the pK values observed in both V/K profiles are the correct ones. Both of the D2O solvent kinetic isotope effects (D2OV=3.8 and D2O(V/Ktrp)=2.8) are pH independent. However, the D2O V/Ktrp decreases below two pK values of 8.1 and 6.5, and the +0.5 .DELTA.pKa indicates that neither of the enzyme groups required for catalysis is a sulfhydryl residue. The primary deuterium isotope effects on V and V/K for tryptophan decrease to 1.25 and 1.82, respectively, when determined in D2O. There is no observable D2O solvent isotope effect on V or V/K for S-methyl-L-cysteine. Thus, internal aldimine formation and aminoacrylate transimination and hydrolysis do not appear to be solvent sensitive, while proton abstraction at the 2-position of the substrate is partially rate-limiting.