Elementary Processes in the Interaction of Tyrosine Phenol Lyase with Inhibitors and Substrate

Muro, Tetsuo; Nakatani, Hiroshi; Kitano, Hiromi; Kumagai, Hidehiko; Yamada, Hideaki

doi:10.1093/oxfordjournals.jbchem.a132168

Cited by 31 publications

(27 citation statements)

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“…Single-wavelength stopped-flow kinetic studies of the reactions of wild-type and [H343A]tyrosine phenol-lyase with L-phenylalanine, L-methionine, L-alanine and L-homoserine were performed at pH 8 and 500 nm. The apparent rate constants, kobs, for the reaction of amino acids to form quinonoid intermediates were found to increase in a hyperbolic manner with the concentration, as reported previously by Muro et al (1978). This behavior in the pre-steady state is diagnostic for a mechanism in which a slow relaxation between the external aldimine and quinonoid complexes is preceded by a rapid binding equilibrium, as shown in Scheme I.…”

Section: Spectra Of Tyrosine Phenol-lyasessupporting

confidence: 79%

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Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

Chen¹,

Gollnick²,

Phillips³

1995

Eur J Biochem

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His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R.L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195--4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indole-lyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for alpha, beta-elimination (except S-ethyl-L-cysteine) exhibited lower kcat (10-30%) and kcat/Km (1-10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for L-phenylalanine and L-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-L-tyrosine with wild-type tyrosine phenol-lyase is identical to that of L-tyrosine described previously [Kiick, D. M. & Phillips, R. S. (1988) Biochemistry 27, 7333-7338]. The pH profile of kcat/Km for this reaction exhibits two pKa values with an average of 7.7 +/- 0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of kcat/Km for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pKa values with an average of 7.8 +/- 0.3. However, kcat for 3-fluoro-L-tyrosine is pH-dependent for the mutant, exhibiting two pKa values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on kcat and kcat/Km for 3-fluoro-L-[alpha-2H]-tyrosine are independent of pH and equal to 3.9 +/- 0.2 and 2.2 +/- 0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4 +/- 0.2 and 3.8 +/- 0.3, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Spectra Of Tyrosine Phenol-lyasessupporting

confidence: 79%

“…Protein was determined by the method of Bradford (1976), with purified tyrosine phenol-lyase as a standard. The concentration of purified tyrosine phenollyase was determined from the absorbance at 278 nm (A'" = 8.37) (Muro et al, 1978) assuming a subunit mass of 51 kDa (Antson et al, 1993).…”

Section: Deae-sephacel Column Chromatographymentioning

confidence: 99%

Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

Chen¹,

Gollnick²,

Phillips³

1995

Eur J Biochem

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“…Mixing of this aminoacrylate complex with indole in the stopped flow spectrophotometer resulted in concentration dependent formation of a quinonoid complex, as expected for an aminoacrylate [37]. This assignment was further confirmed by rapid quench experiments, which demonstrated that indole is formed from L-tryptophan at about 30 s À1 in a stoichiometric burst, followed by the linear steady-state reaction [39]. When benzimidazole is included in stopped-flow reactions of TIL with a-[ 2 H]-L-tryptophan, not only is the expected primary kinetic isotope effect observed on the formation of the quinonoid complex, but also a KIE of 2.99 ± 0.30 is seen on the formation of the aminoacrylate [37].…”

Section: Pre-steady State Kineticssupporting

confidence: 55%

The role of substrate strain in the mechanism of the carbon–carbon lyases

Phillips¹,

Demidkina²,

Faleev³

2014

Bioorganic Chemistry

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“…Previous spectroscopic studies showed that TPL forms a stable quinonoid complex in solution when incubated with L -ala (11,34-36). It was also suggested that a mixture of the external aldimine and the quinonoid intermediate is formed when L -Met is added to TPL in a solution (20) or in crystals (21).…”

Section: Resultsmentioning

confidence: 99%

Insights into the Catalytic Mechanism of Tyrosine Phenol-lyase from X-ray Structures of Quinonoid Intermediates

Milić

Demidkina

Faleev

et al. 2008

Journal of Biological Chemistry

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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.

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Elementary Processes in the Interaction of Tyrosine Phenol Lyase with Inhibitors and Substrate

Cited by 31 publications

References 0 publications

Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

The role of substrate strain in the mechanism of the carbon–carbon lyases

Insights into the Catalytic Mechanism of Tyrosine Phenol-lyase from X-ray Structures of Quinonoid Intermediates

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