Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5'-phosphate during the reaction of L-serine with the alpha 2 beta 2 and beta 2 forms of Escherichia coli tryptophan synthase. In agreement with previous kinetic studies [Lane, A., & Kirschner, K. (1983) Eur. J. Biochem. 129, 561-570], the reaction with alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3). The RSSF data reveal that during tau 1, the internal aldimine, E(PLP), with lambda max = 412 nm (pH 7.8), undergoes rapid conversion to two transient species, one with lambda max congruent to 420 nm and one with lambda max congruent to 460 nm. These species decay in a biphasic process (1/tau 2, 1/tau 3) to a complicated final spectrum with lambda max congruent to 350 nm and with a broad envelope of absorbance extending out to approximately 525 nm. Analysis of the time-resolved spectra establishes that the spectral changes in tau 2 are nearly identical with the spectral changes in tau 3. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to increase the amount of the 420-nm transient and to decrease the amount of the species with lambda max congruent to 460 nm. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with lambda max congruent to 460 nm is the delocalized carbanion (quinoidal) species derived from abstraction of the alpha proton from the external aldimine. The reaction of L-serine with beta 2 consists of two relaxations (1/tau 1 beta greater than 1/tau 2 beta) and yields a quasi-stable species with lambda max = 420 nm, in good agreement with a previous report [Miles, E. W., Hatanaka, M., & Crawford, I. P. (1968) Biochemistry 7, 2742-2753]. Analysis of the RSSF spectra indicates that the same spectral change occurs in each phase of the reaction. The similarity of the spectral changes that occur in tau 2 and tau 3 of the alpha 2 beta 2 reaction is postulated to originate from the existence of two (slowly) interconverting forms of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the reaction of L-serine and indole with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

The pre-steady-state reaction of indole and L-serine with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase has been investigated under different premixing conditions with rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy for the spectral range 300-550 nm. When alpha 2 beta 2 was mixed with indole and L-serine, the reaction of alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3) with rate constants identical with the three relaxations seen in the partial reaction with L-serine [Drewe, W.F., Jr., & Dunn, M.F. (1985) Biochemistry 24, 3977-3987]. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to be similar to the effects observed in the reaction with serine only. The observed spectral changes and isotope effects indicate that the aldimine of L-serine and PLP and the first quinoid derived from this external aldimine are transient species that accumulate during tau 1. Conversion of these intermediates to the alpha-aminoacrylate Schiff base during tau 2 and tau 3 limits the rate of formation of the second quinoidal species (lambda max 476 nm) generated via C-C bond formation between indole and the alpha-aminoacrylate intermediate. The pre-steady-state reaction of the alpha 2 beta 2-serine mixture with indole is comprised of four relaxations (1/tau 1* greater than 1/tau 2* greater than 1/tau 3* greater than 1/tau 4*).(ABSTRACT TRUNCATED AT 250 WORDS)     

Allosteric interactions coordinate catalytic activity between successive metabolic enzymes in the tryptophan synthase bienzyme complex.

Tryptophan synthase from enteric bacteria is an alpha 2 beta 2 bienzyme complex that catalyzes the final two reactions in the biosynthesis of L-tryptophan (L-Trp) from 3-indole-D-glycerol 3'-phosphate (IGP) and L-serine (L-Ser). The bienzyme complex exhibits reciprocal ligand-mediated allosteric interactions between the heterologous subunits [Houben, K., & Dunn, M. F. (1990) Biochemistry 29, 2421-2429], but the relationship between allostery and catalysis had not been completely defined. We have utilized rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy to study the relationship between allostery and catalysis in the alpha beta-reaction catalyzed by the bienzyme complex from Salmonella typhimurium. The pre-steady-state spectral changes that occur when L-Ser and IGP are mixed simultaneously with the alpha 2 beta 2 complex show that IGP binding to the alpha-site accelerates the formation of alpha-aminoacrylate [E(A-A)] from L-Ser at the beta-site. Through the use of L-Ser analogues, we show herein that the formation of the E(A-A) intermediate is the chemical signal which triggers the conformational transition that activates the alpha-subunit. beta-subunit ligands, such as L-Trp, that react to form covalent intermediates at the beta-site, but are incapable of E(A-A) formation, do not stimulate the activity of the alpha-subunit. Titration experiments show that the affinity of G3P and GP at the alpha-site is dependent upon the nature of the chemical intermediate present at the beta-active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton transfers in the beta-reaction catalyzed by tryptophan synthase

Reactions catalyzed by the beta-subunits of the tryptophan synthase alpha(2)beta(2) complex involve multiple covalent transformations facilitated by proton transfers between the coenzyme, the reacting substrates, and acid-base catalytic groups of the enzyme. However, the UV/Vis absorbance spectra of covalent intermediates formed between the pyridoxal 5'-phosphate coenzyme (PLP) and the reacting substrate are remarkably pH-independent. Furthermore, the alpha-aminoacrylate Schiff base intermediate, E(A-A), formed between L-Ser and enzyme-bound PLP has an unusual spectrum with lambda(max) = 350 nm and a shoulder extending to greater than 500 nm. Other PLP enzymes that form E(A-A) species exhibit intense bands with lambda(max) approximately 460-470 nm. To further investigate this unusual tryptophan synthase E(A-A) species, these studies examine the kinetics of H(+) release in the reaction of L-Ser with the enzyme using rapid kinetics and the H(+) indicator phenol red in solutions weakly buffered by substrate L-serine. This work establishes that the reaction of L-Ser with tryptophan synthase gives an H(+) release when the external aldimine of L-Ser, E(Aex(1)), is converted to E(A-A). This same H(+) release occurs in the reaction of L-Ser plus the indole analogue, aniline, in a step that is rate-determining for the appearance of E(Q)(Aniline). We propose that the kinetic and spectroscopic properties of the L-Ser reaction with tryptophan synthase reflect a mechanism wherein the kinetically detected proton release arises from conversion of an E(Aex(1)) species protonated at the Schiff base nitrogen to an E(A-A) species with a neutral Schiff base nitrogen. The mechanistic and conformational implications of this transformation are discussed.     

Aminoacrylate intermediates in the reaction of Citrobacter freundii tyrosine phenol-lyase

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 alpha-carbon to give a quinonoid intermediate. Elimination of phenol then has been proposed to give an alpha-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 alpha-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 alpha-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 approximately 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 alpha-aminoacrylate is observed, which may be due to iminopyruvate formation. Both l-tyrosine and 3-fluoro-l-tyrosine exhibit kinetic isotope effects of approximately 2-3 on alpha-aminoacrylate formation when the alpha-(2)H-labeled substrates are used, consistent with the previously reported internal return of the alpha-proton to the phenol product. These results are the first direct spectroscopic observation of alpha-aminoacrylate intermediates in the reactions of TPL.     

BetaQ114N and betaT110V mutations reveal a critically important role of the substrate alpha-carboxylate site in the reaction specificity of tryptophan synthase

In the PLP-requiring alpha2beta2 tryptophan synthase complex, recognition of the substrate l-Ser at the beta-site includes a loop structure (residues beta110-115) extensively H-bonded to the substrate alpha-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: betaThr110 --> Val, and betaGln114 --> Asn. The betaT110V mutation greatly impairs both catalytic activity in the beta-reaction, and allosteric communication between the alpha- and beta-sites. The crystal structure of the betaT110V mutant shows that the modified l-Ser carboxylate subsite has altered protein interactions that impair beta-site catalysis and the communication of allosteric signals between the alpha- and beta-sites. Purified betaQ114N consists of two species of mutant protein, one with a reddish color (lambdamax = 506 nm). The reddish species is unable to react with l-Ser. The second betaQ114N species displays significant catalytic activities; however, intermediates obtained on reaction with substrate l-Ser and substrate analogues exhibit perturbed UV/vis absorption spectra. Incubation with l-Ser results in the formation of an inactive species during the first 15 min with lambdamax approximately 320 nm, followed by a slower conversion over 24 h to the species with lambdamax = 506 nm. The 320 and 506 nm species originate from conversion of the alpha-aminoacrylate external aldimine to the internal aldimine and alpha-aminoacrylate, followed by the nucleophilic attack of alpha-aminoacrylate on C-4' of the internal aldimine to give a covalent adduct with PLP. Subsequent treatment with sodium hydroxide releases a modified coenzyme consisting of a vinylglyoxylic acid moiety linked through C-4' to the 4-position of the pyridine ring. We conclude that the shortening of the side chain accompanying the replacement of beta114-Gln by Asn relaxes the steric constraints that prevent this reaction in the wild-type enzyme. This study reveals a new layer of structure-function interactions essential for reaction specificity in tryptophan synthase.     

Fluorescence determination of D- and L-tryptophan concentrations in rat plasma following administration of tryptophan enantiomers using HPLC with pre-column derivatization

Similar to L-tryptophan (L-Trp), D-Trp can be converted to unique metabolites in the mammalian body. In the present study, the difference in the plasma half-life (t(1/2)) between Trp enantiomers was investigated by following the alterations in the plasma concentration of D- or L-Trp after intraperitoneal (i.p.) administration of each enantiomer to male Sprague-Dawley rats (100 mg/kg). The investigation was performed using reversed-phase high-performance liquid chromatography (HPLC) and pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS). The t(1/2) value of D-Trp was significantly smaller than that of L-Trp, suggesting that D-Trp was eliminated from the plasma more rapidly than L-Trp. In addition, a significant increase in the plasma concentration of L-Trp was observed following administration of D-Trp, whereas no D-Trp was detected after L-Trp administration. Furthermore, the increase in the plasma concentration of L-Trp was significantly suppressed by pretreatment with an inhibitor of D-amino acid oxidase (DAAO), 3-methylpyrazole-5-carboxylic acid, which suggests that DAAO was involved in the conversion of D-Trp to L-Trp in vivo.     

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole

The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4-2.0 in the reaction of 3-[(2)H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[(2)H]indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm. The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[(2)H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP. These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.     

Detection and identification of transient intermediates in the reactions of tryptophan synthase with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Evidence for a tetrahedral (gem-diamine) intermediate

The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy. The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively). Rate constants determined by SWSF were either well resolved (i.e., k1[DHT], k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1[OXA], k-1 greater than or equal to k2, k-2 greater than k3, k-3). The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species. Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system. The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates.     

Characterization of the allosteric anion-binding site of O-acetylserine sulfhydrylase.

A new crystal structure of the A-isozyme of O-acetylserine sulfhydrylase-A (OASS) with chloride bound to an allosteric site located at the dimer interface has recently been determined [Burkhard, P., Tai, C.-H., Jansonius, J. N., and Cook, P. F. (2000) J. Mol. Biol. 303, 279-286]. Data have been obtained from steady state and presteady-state kinetic studies and from UV-visible spectral studies to characterize the allosteric anion-binding site. Data obtained with chloride and sulfate as inhibitors indicate the following: (i) chloride and sulfate prevent the formation of the external aldimines with L-cysteine or L-serine; (ii) chloride and sulfate increase the external aldimine dissociation constants for O-acetyl-L-serine, L-methionine, and 5-oxo-L-norleucine; (iii) chloride and sulfate bind to the allosteric site in the internal aldimine and alpha-aminoacrylate external aldimine forms of OASS; (iv) sulfate also binds to the active site. Sulfide behaves in a manner identical to chloride and sulfate in preventing the formation of the L-serine external aldimine. The binding of chloride to the allosteric site is pH independent over the pH range 7-9, suggesting no ionizable enzyme side chains ionize over this pH range. Inhibition by sulfide is potent (K(d) is 25 microM at pH 8) suggesting that SH(-) is the physiologic inhibitory species.     

Conversion of D-tryptophan to indole-3-acetic acid in coleoptiles of a normal and a semi-dwarf barley (Hordeum vulgare) strain

Exogenously applied D-tryptophan (D-Trp) was more effective than L-Trp in inducing elongation of coleoptile segments of a normal barley ( Hordeum vulgare L. cv. Akashinriki) strain and a semi-dwarf strain with lower endogenous indole-3-acetic acid (IAA) level. D-cycloserine, an inhibitor of D-aminotransferase, completely inhibited both the D- and L-Trp-induced elongation of both strains. Addition of D-Trp increased IAA levels in both strains 4-fold over endogenous levels. The increase in IAA level was completely inhibited by D-cycloserine. The endogenous L-Trp level of semi-dwarf coleoptiles was similar to that of normal ones. These results suggested that IAA is synthesized by the conversion of L-Trp to indole-3-pyruvic acid via D-Trp in both strains, and that the lower IAA level of the semi-dwarf strain probably is a result of the impeded IAA biosynthesis involved in D-Trp.     

Interaction and combination of separate A and B chains of insulin effects of D-and L-tryptophan in position A1

The S-thiomethyl derivatives of insulin A chain with A1-Gly replaced by D- or L-Trp have been prepared and their respective interaction and combination with the S-thiomethyl B chain studied. The UV difference spectra of the mixed against the separated [Trp1]A chains with the B chain at pH 10.8 are similar to those obtained for the unmodified chains except that the 295-nm-negative peak for ionized Tyr residue appears to be less marked. Fluorescence studies show very little environmental changes at the A1-Trp residues when mixed with the B chain. The intact hormone with A1-Gly replaced by D-Trp is known to be considerably more active than the analog with L-Trp replacement. However, for both derivatives the resynthesis of the whole molecules correctly joined by disulfide bridges starting from the separated reduced chains, gives similar low yields as shown by HPLC analysis and by receptor-binding assay. The replacement of A1-Gly by D-Trp appears to affect the separated A chain more than the intact hormone and replacements at A1 by both D- and L-Trp probably lead to significant conformational changes of the A chain so as to prevent its correct pairing with the B chain.     

Effects of tryptophan and pH on the kinetics of superoxide radical binding to indoleamine 2,3-dioxygenase studied by pulse radiolysis

The reaction of superoxide radical (O2-) with the heme protein indoleamine 2,3-dioxygenase has been investigated by the use of pulse radiolysis. In the absence of the substrate tryptophan (Trp), the ferric enzyme reacted quantitatively with O2- to form the oxygenated enzyme. The rate constant for the reaction (8.0 x 10(6) M-1 s-1 at pH 7.0) increased with a decrease in pH. In the presence of low concentrations of L-Trp (approximately 50 microM), under which the catalytic site of the ferric enzyme is greater than 99% Trp-free at pH 7.0, the only spectral species observed upon O2- binding was L-Trp-bound oxygenated enzyme, the ternary complex. This suggests that under the conditions employed O2- binds first to the ferric enzyme to form the oxygenated enzyme and is followed by rapid binding of L-Trp. It was also found that absorbance changes (delta A) for the enzyme after the pulse were significantly decreased when an increased L-Trp concentration was employed. A 50% decrease in delta A was caused with approximately 50 microM L-Trp at pH 7.0. Similar results were also observed with other indole derivatives with decreasing delta A values in the order of indole, 3-indoleethanol, alpha-methyl-DL-Trp, and D-Trp. These results suggest that there exists a binding site for these compounds in the dioxygenase different from the catalytic site for Trp and, most significantly, that binding of Trp to the effector binding site of the ferric enzyme markedly inhibits its reaction with O2-.     

Comparative study on kynurenic acid production in the rat striatum by tryptophan enantiomers: an in vivo microdialysis study

Kynurenic acid (KYNA), an endogenous antagonist of N-methyl-D-aspartate and α(7) nicotinic acetylcholine receptors, is one of the L-tryptophan (Trp) metabolites. To compare the level of KYNA produced in the striatum of rats after independent administration of L-Trp and D-Trp, rats were intraperitoneally administered L-Trp and/or D-Trp (100 mg/kg), and a microdialysis (MD) probe was implanted in the striatum. The KYNA level in the MD samples was determined using the column-switching high-performance liquid chromatography system. KYNA levels in the MD samples increased by approximately twofold in rats that were administered D-Trp or L-Trp; this result suggests that just as L-Trp, D-Trp was also metabolized to KYNA in the striatum. Additionally, 30 min before the administration of D-Trp, rats were administered 3-methyl pyrazole-5-carboxylic acid (MPC) (50 mg/kg), which is a specific inhibitor of D-amino acid oxidase (DAAO). Pretreatment with MPC suppressed striatal KYNA production; this result suggests that DAAO, encoded by one of the susceptible genes for schizophrenia, may contribute to the production of KYNA from D-Trp in the striatum of rats.     

Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 2. Evidence for the existence of another binding site in the enzyme for indole derivative effectors

To probe the active site of the heme protein indoleamine 2,3-dioxygenase, the effects of 3-indoleethanol (IET) (or tryptophol), one of the known indole derivative effectors, and indole (IND) on the catalytic (Vmax, Km) and spectroscopic properties (optical absorption and CD) of the enzyme were investigated. Assays were performed with the substrate L- or D-tryptophan (Trp) and an ascorbic acid-methylene blue cofactor system at 25 degrees C. This study has shown that, at millimolar concentrations, both IET and IND lower considerably the Km value for D-Trp by approximately 25% and approximately 60%, respectively, at pH 7.0, while neither affects the Km value for L-Trp. Interestingly, however, these effectors exert opposite effects with respect to each other on the Vmax values for both D-Trp and L-Trp: IET enhances the Vmax values by 40-60% while IND lowers them by 12-24%. These effects of IET and IND on the Vmax values may be attributed to the shift in the ferric (inactive) enzyme----ferrous (active) enzyme equilibrium either to the right (IET) or to the left (IND) caused by the binding of these effectors to the enzyme in the steady state of the catalytic reaction. Both effectors induce clearly detectable spectral changes, especially notable in CD spectra, upon binding (in a 1:1 molar ratio, Kd = 10(-4) to 2.5 X 10(-3) M) to the ferrous enzyme and its complexes with O2, CO, and NO, both in the presence and in the absence of L-Trp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystals of tryptophan indole-lyase and tyrosine phenol-lyase form stable quinonoid complexes

The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B., & Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis.     

Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the beta-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium.

In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.     

Beta D305A mutant of tryptophan synthase shows strongly perturbed allosteric regulation and substrate specificity.

Substrate channeling in the tryptophan synthase bienzyme is regulated by allosteric interactions. Allosteric signals are transmitted via a scaffolding of structural elements that includes a monovalent cation-binding site and salt-bridging interactions between the side chains of betaAsp 305, betaArg 141, betaLys 167, and alphaAsp 56 that appear to modulate the interconversion between open and closed conformations. betaAsp 305 also interacts with the hydroxyl group of the substrate L-Ser in some structures. One possible functional role for betaAsp 305 is to ensure the allosteric transmission that triggers the switching of alphabeta-dimeric units between open and closed conformations of low and high activity. This work shows that substitution of betaAsp 305 with Ala (betaD305A) decreases the affinity of the beta-site for the substrate L-Ser, destabilizes the enzyme-bound alpha-aminoacrylate, E(A-A), and quinonoid species, E(Q), and changes the nucleophile specificity of the beta-reaction. The altered specificity provides a biosynthetic route for new L-amino acids derived from substrate analogues. betaD305A also shows an increased rate of formation of pyruvate upon reaction with L-Ser relative to the wild-type enzyme. The formation of pyruvate is strongly inhibited by the binding of benzimidazole to E(A-A). Upon reaction with L-Ser and in the presence of the alpha-site substrate analogue, alpha-glycerol phosphate, the Na(+) form of betaD305A undergoes inactivation via reaction of nascent alpha-aminoacrylate with bound PLP. This work establishes important roles for betaAsp 305 both in the conformational change between open and closed states that takes place at the beta-site during the formation of the E(A-A) and in substrate binding and recognition.     

Interactions of diastereomeric tripeptides of lysyl-5-fluorotryptophyllysine with DNA. 1. Optical and 19F NMR studies of native DNA complexes

Lysyl-5-fluoro-L-tryptophyllysine and lysyl-5-fluoro-D-tryptophyllysine were synthesized, and their interactions with double-stranded DNA were investigated as a model for protein-nucleic acid interactions. The binding to DNA was studied by monitoring various 19F NMR parameters, the fluorescence, and the optical absorbance in thermal denaturation. The 19F resonance of the L-Trp peptide shifts upfield in the presence of DNA, and that of the D-Trp peptide shifts downfield with DNA present. The influence of ionic strength on the binding of each peptide to DNA and the fluorescence quenching titration of each with DNA indicate that electrostatic bonding (approximately 2 per peptide-DNA complex) dominates the binding in each case and accounts for the similar binding constants determined from the fluorescence quenching, i.e., 7.7 X 10(4) M-1 for the L-Trp complex and 6.2 X 10(-1) for the D-Trp complex. The 19F NMR chemical shift, line width, 19F[1H] nuclear Overhauser effect, and spin-lattice relaxation time (T1) changes all indicate that the aromatic moiety of the L-Trp complex, but not that of the D-Trp complex, is stacked between the bases of DNA. The relative increases in DNA melting temperature caused by binding of the tripeptide diastereomers are also consistent with stacking in the case of the L-Trp peptide. The magnitude of the changes and the susceptibility of the 19F NMR chemical shift to altering the solvent isotope (H2O vs. D2O) suggest that the L-Trp ring is not intercalated in the classical sense but is partially inserted between the bases of one strand of the double helix.     

Mechanism of radical-based catalysis in the reaction catalyzed by adenosylcobalamin-dependent ornithine 4,5-aminomutase

We report an analysis of the reaction mechanism of ornithine 4,5-aminomutase, an adenosylcobalamin (AdoCbl)- and pyridoxal L-phosphate (PLP)-dependent enzyme that catalyzes the 1,2-rearrangement of the terminal amino group of D-ornithine to generate (2R,4S)-2,4-diaminopentanoic acid. We show by stopped-flow absorbance studies that binding of the substrate D-ornithine or the substrate analogue D-2,4-diaminobutryic acid (DAB) induces rapid homolysis of the AdoCbl Co-C bond (781 s(-1), D-ornithine; 513 s(-1), DAB). However, only DAB results in the stable formation of a cob(II)alamin species. EPR spectra of DAB and [2,4,4-(2)H(3)]DAB bound to holo-ornithine 4,5-aminomutase suggests strong electronic coupling between cob(II)alamin and a radical form of the substrate analog. Loading of substrate/analogue onto PLP (i.e. formation of an external aldimine) is also rapid (532 s(-1), D-ornithine; 488 s(-1), DAB). In AdoCbl-depleted enzyme, formation of the external aldimine occurs over long time scales (approximately 50 s) and occurs in three resolvable kinetic phases, identifying four distinct spectral intermediates (termed A-D). We infer that these represent the internal aldimine (lambda(max) 416 nm; A), two different unliganded PLP states of the enzyme (lambda(max) at 409 nm; B and C), and the external aldimine (lambda(max) 426 nm; D). An imine linkage with d-ornithine and DAB generates both tautomeric forms of the external aldimine, but with D-ornithine the equilibrium is shifted toward the ketoimine state. The influence of this equilibrium distribution of prototropic isomers in driving homolysis and stabilizing radical intermediate states is discussed. Our work provides the first detailed analysis of radical-based catalysis in this Class III AdoCbl-dependent enzyme.