Resonance Raman spectra of anionic semiquinoid form of a flavoenzyme, D-amino acid oxidase

Resonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1,750 cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1,602 cm-1 band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1,516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4,10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1,331 and 1,292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1,217 and 1,188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with alpha-iminopropionate or N-methyl-alpha-iminopropionate was essentially identical with that of the complex with picolinate.     

Substrate recognition and activation mechanism of D-amino acid oxidase: a study using substrate analogs

We investigated the mechanism of recognition and activation of substrate by D-amino acid oxidase (DAO) by thermodynamical and spectrophotometric methods using zwitterionic ligands [N-methylisonicotinate (NMIN), trigonelline, and homarine] and monoanionic ligands as model compounds of the substrate and the product. In terms of the charge within the substrate D-amino acid, monoanionic (e.g., benzoate), zwitterionic (e.g., NMIN), and dianionic (e.g., terephthalate) ligands are thought to be good models for neutral, basic, and acidic amino acids, respectively, because when a substrate binds to DAO, as previously reported, the a-ammonium group (-NH(3)(+)) probably loses a proton to become neutral (-NH(2)) before the oxidation. Zwitterionic ligands can also be good model compounds of product in the purple complex (the complex of reduced DAO with the product imino acid), because the imino nitrogen of the imino acid is in a protonated cationic form. We also discuss electrostatic interaction, steric effect, and charge-transfer interaction as factors which affect the affinity of substrate/ligand for DAO. Monoanionic ligands have high affinity for neutral forms of oxidized and semiquinoid DAO, while zwitterionic ligands have high affinity for anionic forms of oxidized, semiquinoid, and reduced DAO; this difference was explained by the electrostatic interaction in the active site. The low affinity of homarine (N-methylpicolinate) for oxidized DAO, as in the case of o-methylbenzoate, is due to steric hindrance: one of the ortho carbons of benzoate is near the phenol carbons of Tyr228 and the other ortho carbon is near the carbonyl oxygen of Gly313. The correlation of the affinity of meta- and para-substituted benzoates for oxidized DAO with their Hammet's s values are explained by the HOMO-LUMO interaction between the phenol group of Tyr224 and the benzene ring of benzoate derivative. The pK(a) of neutral flavin [N(3)-H of oxidized flavin, N(5)-H of semiquinoid flavin, and N(1)-H of reduced flavin] decreases by its binding to the apoenzyme. The magnitude of the decrement is oxidized flavin < semiquinoid flavin < reduced flavin. The largest factor in the substantially low pK(a) of reduced flavin in DAO is probably the steric hindrance between the hydrogen atom of H-N(1)(flavin) and the hydrogen atom of H-N of Gly315, which becomes significant when a hydrogen is bound to N(1) of flavin.     

On the ligands in charge-transfer complexes of porcine kidney flavoenzyme D-amino acid oxidase in three redox states: a resonance Raman study.

To investigate the structural modulation of ligands and their interaction in the active-site nanospace when they form charge-transfer (CT) complexes with D-amino acid oxidase (DAO) in three redox states, we compared Raman bands of the ligands in complex with DAO with those of ligands free in solution. Isotope-labeled ligands were synthesized for assignments of observed bands. The COO(-) stretching of ligands observed around, 1,370 cm(-1) downshifted by about 17 cm(-1) upon complexation with oxidized, semiquinoid and reduced DAO, except for the case of reduced DAO-N-methylisonicotinate complex (8 cm(-1) downward shift); the interaction mode of the carboxylate group with the guanidino group of Arg283 and the hydroxy moiety of Tyr228 of DAO is similar in the three redox states. The C=N stretching mode (1,704 cm(-1)) of Delta(1)-piperideine-2-carboxylate (D1PC) downshifted to 1,675 and 1,681 cm(-1) upon complexation with reduced and semiquinoid DAO, respectively. The downward shifts indicate that the C=N bond is weakened upon the complexation. This is probably due mainly to charge-transfer (CT) interaction between D1PC and semiquinoid or reduced flavin, i.e., the partial electron donation from the highest occupied molecular orbital (HOMO) of reduced flavin or a singly occupied molecular orbital (SOMO) of semiquinoid flavin to the lowest unoccupied molecular orbital (LUMO), an antibonding orbital, of D1PC. This speculation was supported by the finding that the magnitude of the shift is smaller by 5 cm(-1) (observed at 1,680 cm(-1)) in the case of reduced DAO reconstituted with 7,8-Cl(2)-FAD, whose reduced form has lower electron-donating ability than natural reduced FAD. The amount of electron flow was estimated by applying the theory of Friedrich and Person [(1966) J. Chem. Phys. 44, 2166-2170] to these complexes; the amounts of charge transfer from reduced FAD and reduced 7,8-Cl(2)-FAD to D1PC were estimated to be about 10 and 8% of one electron, respectively, in the CT complexes of reduced DAO with D1PC.     

Three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase. Optimization of the oxidative half-reaction through alignment of the product with reduced flavin

The three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase (DAO) was solved by cryo-X-ray crystallography; the purple intermediate is known to comprise a complex between the dehydrogenated product, an imino acid, and the reduced form of DAO. The crystalline purple intermediate was obtained by anaerobically soaking crystals of oxidized DAO in a buffer containing excess D-proline as the substrate. The dehydrogenated product, delta(1)-pyrrolidine-2-carboxylate (DPC), is found sandwiched between the phenol ring of Tyr 224 and the planar reduced flavin ring. The cationic protonated imino nitrogen is within hydrogen-bonding distance of the backbone carbonyl oxygen of Gly 313. The carboxyl group of DPC is recognized by the Arg 283 guanidino and Tyr 228 hydroxyl groups through ion-pairing and hydrogen-bonding, respectively. The (+)HN=C double bond of DPC overlaps the N(5)-C(4a) bond of reduced flavin. The electrostatic effect of the cationic nitrogen of DPC is suggested to shift the resonance hybridization of anionic reduced flavin toward a canonical form with a negative charge at C(4a), thereby augmenting the electron density at C(4a), from which electrons are transferred to molecular oxygen during reoxidation of reduced flavin. The reactivity of reduced flavin in the purple intermediate, therefore, is enhanced through the alignment of DPC with respect to reduced flavin.     

Resonance Raman on the purple intermediates of the flavoenzyme D-amino acid oxidase

Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm^?1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm^?1 in the RR spectrum with D-alanine was shifted to 1675 cm^?1 upon [¹⁵N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm^?1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.     

Complex formation between reduced D-amino acid oxidase and pyridine carboxylates

Picolinate binds to a reduced form of D-amino acid oxidase, and the complex formed has a broad absorption band around 600 nm as in the case of the purple intermediate of the enzyme with a substrate. The dissociation constant at 25 degrees C was 35 microns at pH 7.0. The pH dependence (pH 8.3-pH 6.4) of the dissociation constant indicates that one proton is associated with the complex formation, and picolinate protonated at the N atom binds to the reduced enzyme. Resonance Raman spectra of the complex support that picolinate in the complex is a cationic form protonated at the N atom. Nicotinate also binds to the reduced enzyme, but isonicotinate does not.     

A resonance Raman study on a reaction intermediate of Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating).

Resonance Raman (RR) spectra of purple intermediates of L-phenylalanine oxidase (PAO) with non-labeled and isotopically labeled phenylalanines as substrates, i.e., [1-13C], [2-13C], [ring-U-13C6], and [15N]phenylalanines, were measured with excitation at 632.8 nm within the broad absorption band around 540 nm. The spectra obtained resemble those of purple intermediates of D-amino acid oxidase (DAO). The isotope effects on the 1,665 cm-1 band with [15N] or [2-13C]phenylalanine indicate that the band is due to the C = N stretching mode of an imino acid derived from phenylalanine, i.e., alpha-imino-beta-phenylpropionate. The intense band at 1,389 cm-1 is contributed to by the CO2- symmetric stretching and C-CO2- stretching modes of alpha-imino-beta-phenylpropionate. The 1,602 cm-1 band, which does not shift upon isotopic substitution of phenylalanine, corresponds to the 1,605 cm-1 band of DAO purple intermediates and was assigned to a vibrational mode associated with the C(10a) = C(4a) - C(4) = O moiety of reduced flavin. These results confirm that PAO purple intermediates consist of the reduced enzyme and an imino acid derived from a substrate, and suggest that the plane defined by C(10a) = C(4a) - C(4) = O of reduced flavin and the plane containing H2+N = C - CO2- of an imino acid are arranged in close contact to each other, generating a charge-transfer interaction.     

A resonance Raman study on the structures of complexes of flavoprotein D-amino acid oxidase

Resonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is delta 1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-alpha-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2-symmetric stretching mode of the product, such as delta 1-piperideine-2-carboxylate or delta 1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bacterial sarcosine oxidase: identification of novel substrates and a biradical reaction intermediate

Corynebacterial sarcosine oxidase contains both covalently and noncovalently bound FAD and forms complexes with various heterocyclic carboxylic acids (D-proline and 2-furoic, 2-pyrrolecarboxylic, and 2-thiophenecarboxylic acids). 2-Furoic acid, a competitive inhibitor with respect to sarcosine, selectively perturbs the absorption spectrum of the noncovalent flavin, suggesting that the enzyme has a single sarcosine binding site near the noncovalent flavin. Several heterocyclic amines have been identified as new substrates for the enzyme. Similar reactivity is observed with L-proline and L-pipecolic acid whereas L-2-azetidine-carboxylic acid is less reactive. Turnover with L-proline is slow (TN = 4.4 min-1) as compared with sarcosine (TN = 1000 min-1). Anaerobic reduction of the enzyme with heterocyclic amine substrates at pH 8.0 occurs as a biphasic reaction. A similar long-wavelength intermediate is formed in the initial fast phase of each reaction and then decays in a slower second phase to yield 1,5-dihydroFAD. The slow phase is not kinetically significant during aerobic turnover at pH 8.0 and is absent when the anaerobic reactions are conducted at pH 7.0. EPR and other studies at pH 7.0 show that the long-wavelength species is a half-reduced form of the enzyme (1 electron/substrate-reducible flavin) containing 0.9 mol of flavin radical/mol of substrate-reducible flavin. This biradical intermediate exhibits an absorption spectrum similar to that expected for a 50:50 mixture of red anionic and blue neutral flavin radicals. A similar long-wavelength species is observed during titration of the enzyme with sarcosine and other reductants. Studies with L-proline suggest that reduction of the enzyme involves initial transfer of two electrons to the noncovalent flavin. The covalent flavin is not required and can be complexed with sulfite without affecting the rate of electron transfer. The initial half-reduced form of the enzyme appears to be rapidly converted to the biradical form via comproportionation of the reduced noncovalent flavin with the oxidized covalent flavin.     

Proton release during the reductive half-reaction of D-amino acid oxidase

Changes in the net protonation of D-amino acid oxidase during binding of competitive inhibitors and during reduction by amino acids have been monitored using phenol red as a pH indicator. At pH 8.0, no uptake or release of protons from solution occurs upon binding the inhibitors benzoate, anthranilate, picolinate, or L-leucine. The Kd values for both picolinate and anthranilate were determined from pH 5.4 to 9.0. The results are consistent with a single group on the enzyme having a pK of 6.3 which must be unprotonated for tight binding, as is the case with benzoate binding (Quay, S., and Massey, V. (1977) Biochemistry 16, 3348-3354) and with tight binding of the inhibitor form with an unprotonated amino group. Upon reduction of the enzyme by amino acid substrates, two protons are released to solution. The first is released concomitantly with reduction to the reduced enzyme-imino acid charge transfer complex. The second is released only upon dissociation of the charge transfer complex to free reduced enzyme and imino acid. The first proton is assigned as arising from the amino acid group and the second from the amino acid alpha-hydrogen. These results are consistent with the flavin in reduced D-amino acid oxidase being anionic.     

Kinetic and equilibrium studies on the interaction of reduced flavoprotein D-amino acid oxidase with pyridine carboxylates

The equilibrium constants and the rate constants (binding and dissociation constants) between reduced D-amino acid oxidase and pyridine carboxylates were obtained at various pH values (from pH 6.0 to 8.3). The pH dependence of the constants is consistent with the previous conclusion from a resonance Raman study that pyridine carboxylates in the form of a cation protonated at the N atom can bind to the reduced enzyme, but those in the neutral form cannot bind, showing that the positive charge of cationic pyridine carboxylates interacts with the negative charge of the anionic reduced flavin in the reduced enzyme. The binding rate constants of picolinate and nicotinate in the cationic form for the reduced enzyme were quite similar to each other, but the dissociation rate constant of picolinate is several times smaller than that of nicotinate. Thus, it is concluded that the difference in affinity of picolinate and nicotinate for the reduced enzyme is derived from the difference of the dissociation rate constants.     

Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex

The reaction of D-amino acid oxidase [EC 1.4.3.3] (DAO) from porcine kidney with beta-cyano-D-alanine (D-BCNA) was studied. DAO was found to catalyze elimination of the cyano group as well as oxidation of D-BCNA. During the course of the reaction in the presence of excess oxygen, an intermediate was observed which exhibited a characteristic absorption spectrum with a broad charge transfer band in the longer wavelength region. The CD spectrum of this intermediate resembles that of DAO-anthranilate complex. The rate of oxygen consumption in the aerobic reaction decreased with time, suggesting product inhibition due to complex formation between the enzyme and the product. Anaerobic addition of D-BCNA reduced the enzyme to its fully reduced state, the CD spectrum of which closely resembles that of the enzyme reduced by excess D-alanine. When an appropriate amount of D-BCNA was added to the enzyme under air, the charge transfer complex was observed immediately, and underwent a change to the reduced state as the oxygen was consumed. The binding strength in the charge transfer complex was found to be comparable to that in DAO-benzoate complex. The accumulating product in the oxidation of D-BCNA had a strong absorption at 285 nm. The aerobic reaction of beta-cyano-L-alanine (L-BCNA) with snake venom L-amino acid oxidase (LAO) produced the same product with an absorption at 285 nm as the reaction of DAO with D-BCNA. The product obtained in the reaction with LAO was found to form the same charge transfer complex with DAO. We tentatively identified this product as alpha-amino-beta-cyanoacrylate and the charge transfer complex as the complex of alpha-amino-alpha-cyanoacrylate with the oxidized enzyme. A hypothetical reaction pathway based on the present finding is proposed. Addition of L-BCNA to the enzyme produced an absorption spectrum very similar to that of the DAO-benzoate complex without oxidation or elimination. L-BCNA was found to be a competitive inhibitor of the oxidation of D-alanine.     

Molecular characterization of NikD,a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics

Nikkomycin antibiotics are potent inhibitors of chitin synthase, effective as therapeutic antifungal agents in humans and easily degradable insecticides in agriculture. NikD is a novel flavoprotein that catalyzes the oxidation of Delta(1)- or Delta(2)-piperideine-2-carboxylate, a key step in the biosynthesis of nikkomycin antibiotics. The resulting dihydropicolinate product may be further oxidized by nikD or converted to picolinate in a nonenzymic reaction. Saturated nitrogen heterocycles (L-pipecolate, L-proline) and 3,4-dehydro-L-proline act as alternate substrates. The ability of nikD to oxidize 3,4-dehydro-L-proline, but not 1-cyclohexenoate, suggests that the enzyme is specific for the oxidation of a carbon-nitrogen bond. An equivalent reaction is possible with the enamine (Delta(2)), but not the imine (Delta(1)), form of the natural piperideine-2-carboxylate substrate. Apparent steady-state kinetic parameters for the reaction of nikD with Delta(1)- or Delta(2)-piperideine-2-carboxylate (k(cat) = 64 min(-1); K(m) = 5.2 microM) or 3,4-dehydro-L-proline (k(cat) = 18 min(-1); K(m) = 13 mM) were determined in air-saturated buffer by measuring hydrogen peroxide formation in a coupled assay. NikD appears to be a new member of the monomeric sarcosine oxidase (MSOX) family of amine oxidizing enzymes. The enzyme contains 1 mol of flavin adenine dinucleotide (FAD) covalently linked to Cys321. The covalent flavin attachment site and two residues that bind substrate carboxylate in MSOX are conserved in nikD. NikD, however, exhibits an unusual long-wavelength absorption band, attributed to charge-transfer interaction between FAD and an ionizable (pK(a) = 7.3) active-site residue. Similar long-wavelength absorption bands have been observed for flavoproteins containing an active site cysteine or cysteine sulfenic acid. Interestingly, Cys273 in nikD aligns with an active-site histidine in MSOX (His269) that is, otherwise, a highly conserved residue within the MSOX family.     

Proton release from flavoprotein D-amino acid oxidase on complexation with the zwitterionic ligand, trigonelline

Trigonelline, i.e., N-methylnicotinate, which has a zwitterionic structure similar to a substrate D-amino acid, is a useful active site probe for D-amino acid oxidase (DAO). The affinity of trigonelline for DAO in the deprotonated state at the enzyme bound FAD 3-imino group is higher than in the neutral state, contrary to in the case of benzoate, which is a competitive inhibitor and is in a monoanionic form. The time course of the absorbance change was monitored for the binding of DAO with trigonelline by means of a stopped-flow technique. The reaction, on monitoring at 507 nm, was found to be biphasic at pH 8.3, with fast and slow phases. The dissociation of the 3-imino proton of the enzyme bound FAD was observed in the same time course as the slow phase. These results suggest that the positive charge of trigonelline exists near the 3-imino group of the enzyme bound FAD and interacts repulsively with the proton of the 3-imino group. The absorption spectra of the DAO-trigonelline complex at various pHs also support this hypothesis. In the catalysis of DAO, a similar mechanism may be involved, that is, the positive charge of a D-amino acid may interact repulsively with the 3-imino proton of the enzyme bound FAD, and this interaction may be important for the catalysis.     

Polynucleotides. XLVI. 1 Synthesis and properties of poly (2''-amino-2''-deoxyadenylic acid).

Poly (2'-amino-2'-deoxyadenylic acid) [poly (Aa)] was prepared from chemically synthesized 2'-amino-2'-deoxy-ADP by the catalysis of polynucleotide phosphorylase. Poly (Aa) showed a similar UV absorption spectra to poly (A), but quite different CD spectra at pH 7.0 and 5.7. At the former pH it showed a single negative Cotton band and at the latter a curve with a large splitting of bands. Acid titration of poly (Aa) suggested protonated form below pH 7.0. Temperature absorption profiles and their dependency on sodium ion concentration suggested an ordered structure for poly (Aa) which is stabilized by stacking of bases and intrastrand interaction between 2'-amino and internucleotidic phosphate groups. Poly (Aa) forms a 1:2 complex with poly (U) at neutrality and its Tm was 45 degrees in the presence of 0.15M sodium ion.     

Suicide inactivation of the flavoenzyme D-lactate dehydrogenase by alpha-hydroxybutynoate.

The acetylenic alpha-hydroxy acid 2-hydroxy-3-butynoate (alpha HB) is a substrate and an irreversible inactivator of the FAD-containing flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii. On the average, the enzyme undergoes five catalytic turnovers with alpha HB in air at pH 7.0 before being inactivated. Irreversible inactivation is due to the conversion of the flavin to a pink adduct with visible absorption peaks at 522, 382, and 330 nm and weak fluorescence with an emission maximum at 635 nm. The adduct is stable and can be released from the enzyme and purified. It retains a structure analogous to FAD since it binds to the FAD-specific apo-D-amino acid oxidase. It can be further converted to an FMN analogue with phosphodiesterase which binds to the FMN-specific apoflavodoxin. Experiments were conducted to test whether inactivation was initiated by an alpha HB allene carbanion or the dehydrogenation product of alpha HB. Kinetic studies proved inconclusive in that a rapid equilibrium between an oxidized enzyme--allene carbanion pair and reduced enzyme--keto acid pair would make these two species kinetically equivalent. The olefinic substrate 2-hydroxy-3-butenoate, however, produced no flavin adduct. Since the keto acid derived from the oxidation of this alpha-hydroxy acid is expected to be as reactive as 2-keto-3-butynoate, it is concluded that an allene carbanion produced by abstraction of the alpha-hydrogen of alpha HB is the reactive species which covalently adds to the flavin.     

A mobile tryptophan is the intrinsic charge transfer donor in a flavoenzyme essential for nikkomycin antibiotic biosynthesis

The flavoenzyme nikD is required for the biosynthesis of nikkomycin antibiotics. NikD exhibits an unusual long wavelength absorption band attributed to a charge transfer complex of FAD with an unknown charge transfer donor. NikD crystals contain an endogenous active site ligand. At least four different compounds are detected in nikD extracts, including variable amounts of two ADP derivatives that bind to the enzyme's dinucleotide binding motif in competition with FAD, picolinate (0.07 mol/mol of nikD) and an unknown picolinate-like compound. Picolinate, the product of the physiological catalytic reaction, matches the properties deduced for the active site ligand in nikD crystals. The charge transfer band is eliminated upon mixing nikD with excess picolinate but not by a reversible unfolding procedure that removes the picolinate-like compound, ruling out both compounds as the intrinsic charge transfer donor. Mutation of Trp355 to Phe eliminates the charge transfer band, accompanied by a 30-fold decrease in substrate binding affinity. The results provide definitive evidence for Trp355 as the intrinsic charge transfer donor. The indole ring of Trp355 is coplanar with or perpendicular to the flavin ring in "open" or "closed" crystalline forms of nikD, respectively. Importantly, a coplanar configuration is required for charge transfer interaction. Absorption in the long wavelength region therefore constitutes a valuable probe for monitoring conformational changes in solution that are likely to be important in nikD catalysis.     

Both acidic and basic amino acids in an amphitropic enzyme,CTP:phosphocholine cytidylyltransferase,dictate its selectivity for anionic membranes

Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pK(a) that varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo.     

Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase

Monomeric sarcosine oxidase is a flavoenzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine). Rapid reaction kinetic studies under anaerobic conditions at pH 8.0 show that the enzyme forms a charge transfer Michaelis complex with sarcosine (E-FAD(ox).sarcosine) that exhibits an intense long-wavelength absorption band (lambda(max) = 516 nm, epsilon(516) = 4800 M(-)(1) cm(-)(1)). Since charge transfer interaction with sarcosine as donor is possible only with the anionic form of the amino acid, the results indicate that the pK(a) of enzyme-bound sarcosine must be considerably lower than the free amino acid (pK(a) = 10.0). No redox intermediate is detectable during sarcosine oxidation, as judged by the isosbestic spectral course observed for conversion of E-FAD(ox).sarcosine to reduced enzyme at 25 or 5 degrees C. The limiting rate of the reductive half-reaction at 25 degrees C (140 +/- 3 s(-)(1)) is slightly faster than turnover (117 +/- 3 s(-)(1)). The kinetics of formation of the Michaelis charge transfer complex can be directly monitored at 5 degrees C where the reduction rate is 4.5-fold slower and complex stability is increased 2-fold. The observed rate of complex formation exhibits a hyperbolic dependence on sarcosine concentration with a finite Y-intercept, consistent with a mechanism involving formation of an initial complex followed by isomerization to yield a more stable complex. Similar results are obtained for charge transfer complex formation with methylthioacetate. The observed kinetics are consistent with structural studies which show that a conformational change occurs upon binding of methylthioacetate and other competitive inhibitors.