Application of rapid-scanning, stopped-flow spectroscopy to the characterization of intermediates formed in the reactions of L- and D-tryptophan and beta-mercaptoethanol with Escherichia coli tryptophan synthase

The reactions of the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase with D- and L-Trp and the presteady-state reaction of L-Ser and beta-mercaptoethanol under different premixing conditions have been investigated by rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy. The reaction of alpha 2 beta 2 with L-Ser and beta-mercaptoethanol occurs in 3 detectable relaxations with rates similar to the 3 relaxations seen in the partial reaction with L-Ser and in the reaction with L-Ser and indole. The presteady-state phase of the reaction of beta-mercaptoethanol with the alpha-aminoacrylate intermediate is characterized by 2 relaxations. The RSSF spectra for this reaction show that the spectral changes that take place in these 2 phases are essentially identical. The L-Trp reaction is biphasic, and the spectral changes occurring in each phase of the reaction also are identical. The 2 new spectral bands formed (lambda max congruent to 420 nm and congruent to 476 nm) are assigned as the L-Trp external aldimine (Schiff's base) and L-Trp quinonoid intermediates, respectively. The reaction of D-Trp also is biphasic. Analysis of first and second derivatives of the RSSF spectral changes give evidence for the formation of spectral bands with lambda max of approximately 423 nm, approximately 450 nm, and approximately 478 nm. The positions and shapes of these bands suggest a D-Trp external aldimine structure (423 nm) and a quinonoidal species (450 and 478 nm). However, product studies do not support this latter assignment. The behavior of the D- and L-Trp reactions and the reaction of beta-mercaptoethanol with the alpha-aminoacrylate strongly indicate the pre-existence of 2 slowly equilibrating forms of the internal aldimine and of the alpha-aminoacrylate.     

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)     

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.     

Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state

Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.     

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.     

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.     

Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions

Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia. Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.     

L-serine analogues form Schiff base and quinonoidal intermediates with Escherichia coli tryptophan synthase

Substrate analogues of L-serine have been found that react with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase. Upon reaction with alpha 2 beta 2, the analogues glycine, L-histidine, L-alanine, and D-histidine form chemical intermediates derived from reaction with enzyme-bound pyridoxal 5'-phosphate with characteristic UV-visible spectral bands. The spectra of the products of the glycine, L-histidine, and L-alanine reactions with alpha 2 beta 2 contain contributions from the external aldimine, the quinonoid species, and other intermediates along the catalytic pathway. Just as previously reported for the reaction of L-serine with beta 2 [Goldberg, M. E., York, S., & Stryer, L. (1968) Biochemistry 7, 3662-3667], the reactions of glycine, L-histidine, and L-alanine with the beta 2 form of tryptophan synthase yield spectra with no contributions from catalytic intermediates beyond the external aldimine. The kinetics of intermediate formation and comparisons of the time courses for the exchange of alpha-1H for solvent 2H catalyzed by alpha 2 beta 2 or beta 2 were found to be consistent with these assignments. Intermediates further along the tryptophan synthase catalytic pathway are stabilized to a greater degree in the alpha 2 beta 2 complex than in the beta 2 species alone. This observation strongly suggests that the association of alpha and beta subunits to form the native alpha 2 beta 2 species lowers the activation energies for the interconversion of the external aldimine with chemical species further along the catalytic path.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectral tuning of avian violet- and ultraviolet-sensitive visual pigments

The violet- and ultraviolet-sensitive visual pigments of birds belong to the same class of pigments as the violet-sensitive (so-called blue) pigments of mammals. However, unlike the pigments from mammals and other vertebrate taxa which, depending on species, have lambda(max) values of either around 430 nm or around 370 nm, avian pigments are found with lambda(max) values spread across this range. In this paper, we present the sequences of two pigments isolated from Humbolt penguin and pigeon with intermediate lambda(max) values of 403 and 409 nm, respectively. By comparing the amino acid sequences of these pigments with the true UV pigments of budgerigar and canary and with chicken violet with a lambda(max) value of 420 nm, we have been able to identify five amino acid sites that show a pattern of substitution between species that is consistent with differences in lambda(max). Each of these substitutions has been introduced into budgerigar cDNA and expressed in vitro in COS-7 cells. Only three resulted in spectral shifts in the regenerated pigment; two had relatively small effects and may account for the spectral shifts between penguin, pigeon, and chicken whereas one, the replacement of Ser by Cys at site 90 in the UV pigments, produced a 35 nm shortwave shift that could account for the spectral shift from 403 nm in penguin to around 370 nm in budgerigar and canary.     

L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase

L-Vinylglycine (L-VG) has been shown to be a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) synthase [Satoh, S., and Yang, S. F. (1989) Plant Physiol. 91, 1036-1039] as well as of other pyridoxal phosphate-dependent enzymes. This report demonstrates that L-VG is primarily an alternative substrate for the enzyme. The L-VG deaminase activity of ACC synthase yields the products alpha-ketobutyrate and ammonia with a k(cat) value of 1.8 s(-1) and a K(m) value of 1.4 mM. The k(cat)/K(m) of 1300 M(-1) s(-1) is 0.17% that of the diffusion-controlled reaction with the preferred substrate, S-adenosyl-L-methionine. The enzyme-L-VG complex partitions to products 500 times for every inactivation event. The catalytic mechanism proceeds through a spectrophotometrically detected quinonoid with lambda(max) of 530 nm, which must rearrange to a 2-aminocrotonate aldimine to yield final products. Alternative mechanisms for the inactivation reaction are presented, and the observed kinetics for the full reaction course are satisfactorily modeled by kinetic simulation. The inactive enzyme is an aldimine with lambda(max) of 432 nm. It is resistant to NaBH(3)CN but is reduced by NaBH(4). ACC synthase is now expressed in Pichia pastoris with an improved yield of 10 mg/L.     

Fluorescence studies of the interaction of DNA with Hoechst 33258

Absorption and fluorescence measurements of DNA-Hoechst 33258 complexes at high molar ratio of DNA phosphate to dye are consistent with the existence of two types of bound species. One type (Type I) predominates at high ionic strength, whereas the other (Type II) occurs at low ionic strength. The fluorescence peak (lambda fmax) depends on the excitation wavelength (lambda ex); lambda fmax shifts toward longer wavelength with increasing lambda ex. Optical properties obtained are summarized in the following: for Type I, lambda amax (absorption) = 352 nm, lambda fmax at lambda ex of 335 nm = 460 nm, tau (fluorescence lifetime) = 2.0-2.5 ns; for Type II, lambda amax = 360 nm, lambda fmax at lambda ex of 335 nm = 470 nm, tau = 4.0-5.0 ns. This behavior is interpreted in terms of solvent-solute relaxation. Type I corresponds to less hydrated bound species, while Type II to more hydrated bound species.     

Kinetic and structural insight into the mechanism of BphD, a C-C bond hydrolase from the biphenyl degradation pathway

Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD(LB400)) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 degrees C showed that the enzyme rapidly (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm) into a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately k(cat)) with simultaneous biphasic appearance (48 and 8 s(-1)) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/tau(1) and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(3) represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and, therefore, postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 A resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.     

Absorption spectra of radical forms of 2,4-dihydroxybenzoic acid, a substrate for p-hydroxybenzoate hydroxylase.

Combined optical and conductimetric measurements in aqueous solution indicate that at high pH (greater than or equal to 10).OH radicals react with the phenoxide form of 2,4-dihydroxybenzoic acid to form transiently phenoxyl radicals and a small amount of hydroxyeyclohexadienyl (HCHD) radicals by 150 ns. The respective yields of 88 and 12% of the total.OH radical yield were deduced from conductance and optical changes as well as from studies using a low potential reductant. The HCHD radical possesses a pKa of 8.0 +/- 0.1 and the constructed spectrum of the deprotonated forms of HCHD has a lambda max at 420 nm with a minimum extinction coefficient of approximately 7250 M-1 cm-1. The red shift in lambda max and increase in extinction coefficient compared to the revised spectral properties of the protonated form of the HCHD radical (lambda max at 390 nm with extinction coefficient of approximately 4500 M-1 cm-1), together with the pKa of the HCHD radical, provide an explanation for the pH-dependent spectral changes of the so-called highly absorbing intermediate II species, observed in the functioning of the enzyme p-hydroxybenzoate hydroxylase. These results add further to the evidence in support of the proposal that intermediate II is composed of species which absorb similarly to the flavin 4(a)-hydroxide and a form of the substrate/product such as the HCHD radical (Anderson, R. F., Patel, K. B., and Stratford, M. R. L. (1987) J. Biol. Chem. 262, 17475-17479).     

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.     

Isolation and characterization of a subunit form of the light-harvesting complex of Rhodospirillum rubrum

A new method is described for the isolation of subunits of the light-harvesting complex from Rhodospirillum rubrum (wild type and the G-9 mutant) in yields that approach 100%. The procedure involved treating membrane vesicles with ethylenediaminetetraacetic acid-Triton X-100 to remove components other than the light-harvesting complex and reaction center. In the preparation from wild-type cells, a benzene extraction was then employed to remove carotenoid and ubiquinone. The next step involved a careful addition of the detergent n-octyl beta-D-glucopyranoside, which resulted in a quantitative shift of the long-wavelength absorbance maximum from 873 to 820 nm. This latter complex was then separated from reaction centers by gel filtration on Sephadex G-100. The pigment-protein complex, now absorbing at 820 nm, contained two polypeptides of about 6-kilodalton molecular mass (referred to as alpha and beta) in a 1:1 ratio and two molecules of bacteriochlorophyll (BChl) for each alpha beta pair. This complex is much smaller in size than the original complex absorbing at 873 nm but probably is an associated form such as alpha 2 beta 2 X 4BChl or alpha 3 beta 3 X 6BChl. The 820-nm form could be completely shifted back to a form once again having a longer wavelength lambda max near 873 nm by decreasing the octyl glucoside concentration. Thus, the complex absorbing at 820 nm appears to be a subunit form of the original 873-nm complex.     

Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.     

Visualization of PLP-bound intermediates in hemeless variants of human cystathionine beta-synthase: evidence that lysine 119 is a general base

Cystathionine beta-synthase catalyzes the condensation of serine and homocysteine to give cystathionine in a pyridoxal phosphate (PLP)-dependent reaction. The human enzyme contains a single heme per monomer that is bound in an N-terminal 69 amino acid extension that is missing from the otherwise highly homologous yeast enzyme. The heme dominates the UV-visible spectrum and obscures kinetic characterization of the PLP-bound reaction intermediates. In this study, we have engineered a hemeless mutant of human cystathionine beta-synthase by deletion of the N-terminal 69 amino acids. The resulting variant displays approximately 40% of the activity seen with the wild type enzyme, binds stoichiometric amounts of PLP, and permits spectral characterization of PLP-based intermediates. The enzyme as isolated exhibits an absorption maximum at 412nm corresponding to a protonated internal aldimine. Addition of serine shifts the lambdamax to 420nm (assigned as the external aldimine) with a broad shoulder between 450 and 500nm (assigned as the aminoacrylate intermediate). Addition of the product, cystathionine, also leads to formation of an external aldimine (420nm). Homocysteine elicits a red shift (and a decrease in absorption) in the spectrum from 412 to 424nm and an increase in absorption at 330nm, presumably due to formation of a dead-end complex. Mutation of K119, the residue that forms the Schiff base, to alanine results in a approximately 10(3)-fold decrease in activity, which increases approximately 2-fold in the presence of an exogenous base, ethylamine. Spectral shifts (412 --> 420nm) consistent with the formation of external aldimines are observed in the presence of serine or cystathionine, but an aminoacrylate intermediate is not formed at detectable levels. These results are consistent with an additional role for K119 as a general base in the reaction catalyzed by human cystathionine beta-synthase.     

Similarity of decay-associated spectra for tryptophan fluorescence of proteins with different structures

Tryptophan fluorescence lifetimes were analyzed for three proteins: human serum albumin, bovine serum albumin, and bacterial luciferase, which contain one, two, and seven tryptophan residues, respectively. For all of the proteins, the fluorescence decays were fitted by three lifetimes: τ₁ = 6–7 ns, τ₂ = 2.0–2.3 ns, and τ₃ ≤ 0.1 ns (the native state), and τ₁ = 4.4–4.6 ns, τ₂ = 1.7–1.8 ns, and τ₃ ≤ 0.1 ns (the denatured state). Corresponding decay-associated spectra had similar peak wavelengths and spectrum half-widths both in the native state (\(\lambda _{\max }^{{\tau _1}} = 324nm\), \(\lambda _{\max }^{{\tau _2}} = 328nm\), and \(\lambda _{\max }^{{\tau _3}} = 315nm\)), and in the denatured state (\(\lambda _{\max }^{{\tau _1}} = 350nm\), \(\lambda _{\max }^{{\tau _2}} = 343nm\), and \(\lambda _{\max }^{{\tau _3}} = 317nm\)). The differences in the steady-state spectra of the studied proteins were accounted for the individual ratio of the lifetime component contributions. The lifetime components were compared with a classification of tryptophan residues in the structure of these proteins within the discrete states model.     

Eye function of Mysidacea (Crustacea) in the northern Baltic Sea

Eye spectral sensitivity, [S(lambda)], was measured in seven northern Baltic mysid species using an electroretinogram technique. Their S(lambda) curves were compared with the spectral distribution of underwater light at their normal habitats. In the littoral species Neomysis integer, Praunus flexuosus and Praunus inermis, the S(lambda) maxima, [S(lambda)(max)], were in the wavelength-bands of 525-535, 505-515 and 520-530 nm respectively. The neoimmigrant species Hemimysis anomala had a S(lambda)(max) around 500 nm and high sensitivity at 393 nm, possibly indicating UV-sensitivity. S(lambda) of the pelagic species Mysis mixta and Mysis relicta sp. II was at about 505-520 nm. M. relicta sp. I from Pojoviken Bay and fresh water humic Lake P??j?rvi had S(lambda)(max) at approximately 550 nm and 570 nm respectively. This is in accordance with a similar long-wavelength shift in light transmittance of the respective waters. The eyes of the latter population were also damaged by strong light. The pontocaspian neoimmigrant H. anomala is clearly adapted to waters transmitting more blue light.     

Characterization of recombinant diaminopropionate ammonia-lyase from Escherichia coli and Salmonella typhimurium

Diaminopropionate ammonia-lyase gene from Escherichia coli and Salmonella typhimurium was cloned and the overexpressed enzymes were purified to homogeneity. The k(cat) values, determined for the recombinant enzymes with DL-DAP, D-serine, and L-serine as substrates, showed that the enzyme from S. typhimurium was more active than that from E. coli and the K(m) values were found to be similar. The purified enzymes had an absorption maximum (lambda(max)) at 412 nm, typical of PLP dependent enzymes. A red shift in lambda(max) was observed immediately after the addition of 10mM DL-DAP, which returned to the original lambda(max) of 412 nm in about 4 min. This red shift might reflect the formation of an external aldimine and/or other transient intermediates of the reaction. The apoenzyme of E. coli and S. typhimurium prepared by treatment with L-cysteine could be partially (60%) reconstituted by the addition of PLP. The holo, apo, and the reconstituted enzymes were shown to be present as homo dimers by size exclusion chromatography.