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Report is presented on the binary complex structures of enzyme PrnB with D and L-tryptophan and D and L-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with the tryptophan 2,3-dioxygenase crystal structures.

The interaction of H463F mutant Escherichia coli tryptophan indole-lyase (H463F Trpase) with competitive inhibitor L-Trp and L-Met is examined. It is seen that both l-Trp and L-Met bind to H463F Trpase to form equilibrating mixtures of external aldimine and quinonoid intermediates but there is a contribution to the deprotonation reaction either from quantum mechanical tunneling or from a mechanical coupling of protein motion and proton transfer in reactions with L-Trp but not with L-Met.

Fluorescence emission spectroscopy was used to investigate interactions between two effectors and BenM, a transcriptional regulator of benzoate catabolism. BenM had a higher affinity for cis,cis-muconate than for benzoate as the sole effector. However, the presence of benzoate increased the apparent dissociation constant (reduced the affinity) of the protein for cis,cis-muconate. Similar results were obtained with truncated BenM lacking the DNA-binding domain. High-level transcriptional activation may require that some monomers within a BenM tetramer bind benzoate and others bind cis,cis-muconate.

Research has been conducted on superoxide reductases from Desulfovibrio vulgaris containing square pyramidal ferrous site which reduces superoxide to hydrogen peroxide. The results of the pulse radiolysis studies carried out in investigating recombinant two-iron superoxide reductase from D. vulgaris are reported.

Research has been conducted on Escherichia coli tryptophan indole-lyase. The preparation of the mutant forms of the indole-lyase carried out in determining the role of these residues in the catalytic mechanism and in reaction specificity for L-tryptophan is reported.

Research has been conducted on the 2-azatyrosine and 3-azatyrosine. The interactions of these enzymes with the tyrosine phenol-lyase from Citrobacter freundii have been investigated and the results are reported.

The effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) in Citrobacter freundii has been studied to find out what the role of Glu69 is in activation of tyrosine phenol-lyase (TPL). Inhibition of wild-type TPL caused by high concentrations of all monovalent cations is likely due to cation binding to the ES complex to form a complex that releases pyruvate slowly.

A conserved O(sub.2) binding pocket residue in Phascolopsis gouldii myohemerythrin (myoHr), L104, has been mutated to several other residues, and study was carried out to look at effects on O(sub.2) association and dissociation rates, O(sub.2) affinity, and autoxidation. L104 was seen to give a hydrophobic barrier that restricts water entry to the oxymyoHr binding pocket. A leucine at position 104 in myoHr thus seems to have the best size/hydrophobicity to help O(sub.2) binding while at the same time opposing autoxidation.

Position 5 of the indole ring of tryptophan appears to play an important role in the formation of 5-hydroxytryptophan from tryptophan catalyzed by tryptophan hydroxylase. Oxygen transfer to the amino acid seems to be the rate-limiting step in the reaction.

Results of an exploratory research on the effect of alteration of the pK(sub a) of Y14 hydroxyl on the kinetic constants of the Y55F/Y88F form of the delta(super 5)-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni, EC 5.3.3.1, are presented. Results indicate that 3-fluorotyrosine is incorporated at high efficiency in place of tyrosine-14 and that 2-fluorotyrosine and 2,3-difluorotyrosine can be substituted at high efficiency. The steady-state kinetic characteristics of Y55K/Y88F KSI and its fluorotyrosine modified forms were determined.

A study was conducted to analyze the reaction of Pseudomonas fluorescens kynureninase with L-kynurenine and L-alanine using rapid-scanning stopped-flow spectrophotometry. A Cary 1E UV/visible spectrophotometer was used to determine steady-state kinetic measurements while rapid chemical quench studies were carried out using a KinTek Instrument apparatus. Results showed that quinonoid and ketimine intermediates support a kynureninase mechanism.

A study was conducted on the effects of pH and isotopic substitution of substrate and solvent on the reaction of kynureninase from Pseudomonas fluorescens. The findings indicate that the rate-limiting step in the reaction of kynureninase occurring subsequent to the first irreversible step, which is anthranilate release, is base catalyzed and involves a single-proton transfer. Thus, the rate-limiting step in the aforementioned reaction is C-4' deprotonation of the pyruvate pyridoxamine 5'-phosphate ketimine intermediate.

X-ray crystallographic studies of Citrobacter freundii tyrosine phenol-lyase (TPL) complexed with the substrate analog 3-(4'-hydroxyphenyl)propionic acid, site directed mutagenesis and kinetic analysis were used to determine the role of Arginine 381 in TPL catalysis. Results indicate that Arg 381 is the only group located in front of the phenolic OH of the bound substrate analog. Furthermore, results suggest that Arg 381 is responsible for the substrate specificity of TPL.

The reaction of the tryptophan synthase-catalyzed aza and thia analogs of L-trytophan with Escherichia coli trytophan indole-lyase were analyzed by rapid-scanning stopped-flow kinetic methods. The beta-elimination reaction between the thia and aza analogs and indole lyase involves a slow internal proton transfer from C-alpha to C-3 of the substrate indole ring. Furthermore, the slow C-protonation of the azaindole rings of the analogs mediates the slow rate-determining proton transfer in the substrate indole ring.

The reaction of Citrobacter freundii tyrosine phenol-lyase with L- as well as d-alanine were investigated. The results reveal the discovery of separate quinoid intermediates and the initial assessment of the total rate constants for alanine racemization in which tyrosine was the catalyst. These data give a basis for future experiments on the alpha,beta-excluding interaction.

The three dimensional structure of tyrosine phenol-lyase from Citrobacter freundii was studied using cloning techniques and amino acid sequence analysis. Five peptides were purified and conformed to sequence derived from gene structure. Radiation diffraction studies were then performed on the crystal structure of tyrosine phenol-lyase. The molecule was analyzed for symmetry, binding sites and biologic activity of subunits. Similarities were noted with the pyridoxal 5-phosphate binding site of aminotransferases and the binding domain showed conservation of active amino acid residues.

A series of aryl-substituted [beta]-benzoyl-DL-alanines was synthesized to evaluate the substrate activity with kynureninase from Pseudomonas fluorescens. Rapid-scanning stopped-flow kinetic experiments demonstrated that substituents have relatively minor effects on formation of the quinonoid and 348 nm intermediates but have a much greater effect on the formation of the aldol product from reaction of benzaldehyde with the 348 nm intermediate.

The enantiomers of a series of phenyl ring-containing secondary alcohols are produced by asymmetric reduction and enantioselective oxidation via kinetic resolution (KR) by using Thermoanaerobacter ethanolicus (W110A TESADH). W110A TESADH in Tris buffer/acetone is used to obtain (R)-alcohols, the anti-Prelog products, by enantiospecific oxidation of (S)-alcohols through oxidative kinetic resolution of the rac-alcohols.

The steady-state and pre-steady-state kinetics of alanine racemization catalyzed by tyrosine phenol-lyase is studied and the effect of phenol and analogues is investigated on this reaction. The results show that there are two distinct quinonoid intermediates formed from either D- or L-alanine and suggest that interconversion of quinonoid intermediates is rate-limiting in the racernization of alanine.

The effect of hydrostatic pressure on the tryptophan (Trp) synthase alpha2beta2 complex from Salmonella typhimurium is investigated. The equilibrium between the open and closed conformations of Trp synthase is found to be affected by a wide range of variables, including alpha-subunit ligands, monovalent cations, organic solvents, pH, and temperature.

K25A, K256H, K256R, and E69D/K256R mutant tyrosine phenol-lyase (TPLs) are prepared to probe the role of Lys-256 in monovalent cation binding and activation and binding. Results show that the function of Lys-256 in TPL is monovalent cation binding and activation.

The pre-steady-state kinetics of the reaction of Beta-benzoyl-L-alanine was investigated by rapid scanning stopped-flow spectrophotometry. The spectra show the formation of a quinonoid intermediate and also support the formation of benzoate as the rate-determining step.

Kynureninase is made up of pyridoxal-5'-phosphate [PLP}-dependent enzyme that catalyzes the hydrolytic cleavage of L-kynurenine to anthranilic acid and L-alanine is discussed. The structure of Pseudomonas fluorescens kynureninase gives information of the role of active site residues in the mechanism of the reaction using site-directed mutagenesis.

Results indicate that aspartate in anthranilate 1,2-dioxygenase maintains the protonated state and reduction potential of the Rieske center but does not play a role in a facile intersite electron transfer pathway or in substrate gating. Data suggest the residuemay be critical for substrate dihydroxylation at or near the mononuclear iron site.

Results point out that the catalysis of the reversible hydrolytic cleavage of L-tryptophan by the tryptophan indole-lyase from Proteus vulgaris is mediated by amino acid residues aspartate-133 and histidine-458. Data indicate that the residues participate in the elimination reaction of L-tryptophan with aspartate-133 forming a hydrogen bond with the indole NH of the substrate and histidine-458 hydrogen bonded to aspartate-133.