The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the Cβ–Cγ bond of l-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon–carbon bond. As for most other pyridoxal 5′-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-l-tyrosine followed by flash-cooling. The X-ray structures, determined at ∼2.0 Å resolution, reveal two quinonoid geometries: “relaxed” in the open and “tense” in the closed state of the active site. The “tense” state is characterized by changes in enzyme contacts made with the substrate’s phenolic moiety, which result in significantly strained conformation at Cβ and Cγ positions. We also captured, at 2.25 Å resolution, the X-ray structure for the state just after the substrate’s Cβ–Cγ bond cleavage by preparing the ternary complex between TPL, alanine quinonoid and pyridine N-oxide, which mimics the α-aminoacrylate intermediate with bound phenol. In this state, the enzyme–ligand contacts remain almost exactly the same as in the “tense” quinonoid, indicating that the strain induced by the closure of the active site facilitates elimination of phenol. Taken together, structural observations demonstrate that the enzyme serves not only to stabilize the transition state but also to destabilize the ground state.

Amino acid transformations catalyzed by a number of PLP-dependent enzymes involve abstraction of the Cα proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. In spite of the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9 Å and 1.95 Å resolution, respectively, by cryocrystallography. The data reveal a network of protein–PLP–substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations – open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the β-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Cα proton.

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is a pyridoxal 5′-phosphate (PLP)dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Tyr to give phenol and ammonium pyruvate. The proposed reaction mechanism for TPL involves formation of an external aldimine of the substrate, followed by deprotonation of the R-carbon to give a quinonoid intermediate. Elimination of phenol then has been proposed to give an R-aminoacrylate Schiff base, which releases iminopyruvate that ultimately undergoes hydrolysis to yield ammonium pyruvate. Previous stopped-flow kinetic experiments have provided direct spectroscopic evidence for the formation of the external aldimine and quinonoid intermediates in the reactions of substrates and inhibitors; however, the predicted R-aminoacrylate intermediate has not been previously observed. We have found that 4-hydroxypyridine, a non-nucleophilic analogue of phenol, selectively binds and stabilizes aminoacrylate intermediates in reactions of TPL with S-alkyl-L-cysteines, L-tyrosine, and 3-fluoro-L-tyrosine. In the presence of 4-hydroxypyridine, a new absorption band at 338 nm, assigned to the R-aminoacrylate, is observed with these substrates. Formation of the 338 nm peaks is concomitant with the decay of the quinonoid intermediates, with good isosbestic points at ∼365 nm. The value of the rate constant for aminoacrylate formation is similar to k cat , suggesting that leaving group elimination is at least partially rate limiting in TPL reactions. In the reaction of S-ethyl-L-cysteine in the presence of 4-hydroxypyridine, a subsequent slow reaction of the R-aminoacrylate is observed, which may be due to iminopyruvate formation. Both L-tyrosine and 3-fluoro-L-tyrosine exhibit kinetic isotope effects of ∼2-3 on R-aminoacrylate formation when the R-2 H-labeled substrates are used, consistent with the previously reported internal return of the R-proton to the phenol product. These results are the first direct spectroscopic observation of R-aminoacrylate intermediates in the reactions of TPL.

Thr-124 and Phe-448 are located in the active site of Citrobacter freundii tyrosine phenol-lyase (TPL) near the phenol ring of a bound substrate analogue, 3-(4'-hydroxyphenyl)propionic acid [Sundararaju, Antson, Phillips, Demidkina, Barbolina, Gollnick, Dodson and Wilson (1997) Biochemistry 36, 6502-6510]. Thr-124 is replaced by Asp and Phe-448 is replaced by His in the crystal structure of a structurally similar enzyme, Proteus vulgaris tryptophan indole-lyase, which has 50% identical residues. Hence, Thr-124 and Phe-448 in TPL were mutated to Ala or Asp, and His, respectively, in order to probe the role of these residues in the reaction specificity for L-Tyr. These mutant enzymes have little or no beta-elimination activity with L-Tyr or 3-fluoro-L-Tyr as a substrate, but retain significant elimination activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines and beta-chloroalanine. Furthermore, the binding of L-Tyr and other non-substrate amino acids is not significantly affected by the mutations. The mutant TPLs form intermediates in rapid-scanning stopped-flow experiments with L-Phe, L-Tyr and L-Trp, similar to those seen with wild-type TPL. These results demonstrate that Thr-124 and Phe-448 are necessary for the reaction specificity of TPL for L-Tyr, and probably play a role in the elimination stage of the reaction mechanism. Thr-124 is within hydrogen-bonding distance of the phenolic group of the bound substrate, and may help to orientate the ring for beta-elimination to occur. Phe-448 may be important to allow the formation of the closed conformation during the reaction.

No abstract

Tryptophan indole-lyase from Escherichia coli catalyzes the reversible cleavage of L-tryptophan to indole and ammonium pyruvate. This reaction is mechanistically interesting since it involves the elimination of an aromatic carbon leaving group. We have been studying the mechanism of tryptophan indole-lyase using rapid-scanning stopped-flow spectrophotometry. Recently, we demonstrated that the rate constant for R-aminoacrylate intermediate formation from R-2 H-L-tryptophan exhibits an isotope effect of 3.0 (Sloan, M. J.; Phillips, R. S. Biochemistry 1996, 35, 16165-16173). We have confirmed this previous result ( D k ) 2.99 ( 0.30) and we have now found that , -di-2 H-L-tryptophan also exhibits a secondary isotope effect ( D k ) 1.17 ( 0.03) on the elimination reaction. Furthermore, R, , -tri-2 H-L-tryptophan exhibits a multiple isotope effect ( D k ) 4.42 ( 0.67) on the elimination of indole. In addition, there is a significant solvent isotope effect ( D k ) 1.79 ( 0.11) on indole elimination in D 2 O. This solvent isotope effect combines with the effect of R-deuterium, since elimination of R-2 H-L-tryptophan in D 2 O exhibits D k ) 4.30 ( 0.16. In addition, the rate constant for indole elimination shows a linear Eyring plot between 5 and 35°C. In the direction of tryptophan synthesis, the reaction of the R-aminoacrylate intermediate with indole to form a quinonoid intermediate also exhibits a kinetic isotope effect for 3-2 H-indole, with D k ) 1.88 ( 0.19. In contrast to our expectations, the results suggest that the proton transfer and carbon-carbon bond cleavage in the elimination reaction are very nearly simultaneous and that the indolenine structure is a transient intermediate which occupies a very shallow well on the reaction coordinate, or a transition state, in the reaction of Trpase.

No abstract

Kinetic parameters of Citrobacter freundii methionine γ-lyase were determined with substrates in γ-elimination reactions as well as the inhibition of the enzyme in the γ-elimination of L-methionine by amino acids with different structure. The data indicate an important contribution of the sulfur atom and methylene groups to the efficiency of binding of substrates and inhibitors. The rate constants of the enzyme-catalyzed exchange of C-α- and C-β-protons with deuterium were determined, as well as the kinetic isotope effect of the deuterium label in the C-α-position of inhibitors on the rate of exchange of their β-protons. Neither stereoselectivity in the β-proton exchange nor noticeable α-isotope effect on the exchange rates of β-protons was found. The ionic and tautomeric composition of the external Schiff base of methionine γ-lyase was determined. Spectral characteristics (absorption and circular dichroism spectra) of complexes with substrates and inhibitors were determined. The spectral and kinetic data indicate that deamination of aminocrotonate should be the rate-determining stage of the enzymatic reaction.