The amino acid sequences of the flavin-peptides of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria

The flavoenzymes dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) contain covalently bound FAD linked via the 8 alpha-position of the isoalloxazine ring to the imidazole N(3) of a histidine residue (Cook, R. J., Misono, K. S., and Wagner, C. (1984) J. Biol. Chem. 259, 12475-12480). The flavin-peptides from tryptic digests of these two enzymes have been isolated and sequenced. Automated sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase contained 25 amino acid residues in the following sequence: Ser-Glu-Leu-Thr-Ala-Gly-Ser- Thr-Trp-His(flavin)-Ala-Ala-Gly-Leu-Thr-Thr-Tyr-Phe-His-Pro-Gly-Ile-A sn-Leu-Lys. The sequence determined for the flavin-peptide from sarcosine dehydrogenase contained 14 amino acid residues Leu-Thr-Ser-Gly-Thr-Thr-Trp-His(flavin)-Thr-Ala-Gly-Leu-Gly-Arg.     

Chromatography and Atomic Absorption Spectrometry for the Assessment of Heavy Metal Distribution among Amino Acids Used in Parenteral Nutrition Formulations—Studies with Cadmium and Lead

The distribution of Cd (II) and Pb (II) among amino acids in parenteral nutrition formulations was investigated by coupling ion-chromatography (HPLC/IC) and electrothermal atomic absorption spectrometry. The methodology was based on ion-exchange separation and fluorimetric amino acid detection after post-column derivatization. Cd (II) and Pb (II) were assayed in 500-µL fractions of the column effluent. The distribution of Cd (II) and Pb (II) in alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), histidine (His), methionine (Met), phenylalanine (Phe), serine (Ser), and threonine (Thr) were analyzed by monitoring changes in the concentration of free amino acids by HPLC/IC. The results indicated that Cd (II) and Pb (II) were distributed according to the following trend: Gly–Cd?>?Gly–Pb?>?Ala–Cd?>?Ala–Pb?>?His–Cd?～?His–Pb?>?Thr–Cd?>?Thr–Pb?>?Phe–Cd?～?Phe–Pb?～?Asp–Cd?～?Asp–Pb?～?Met–Cd?～?Met–Pb?～?Glu–Cd?～?Glu–Pb?>?Ser–Cd?～?Ser–Pb. The effects of amino acid concentration and stability constants of amino acid–metal complexes are discussed.     

Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000- to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0- or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme.sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 x 10(5) M(-1) s(-1)) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.     

Role of the covalent flavin linkage in monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a prototypical member of a recently recognized family of amine-oxidizing enzymes that all contain covalently bound flavin. Mutation of the covalent flavin attachment site in MSOX produces a catalytically inactive apoprotein (apoCys315Ala) that forms an unstable complex with FAD (K(d) = 100 muM), similar to that observed with wild-type apoMSOX where the complex is formed as an intermediate during covalent flavin attachment. In situ reconstitution of sarcosine oxidase activity is achieved by assaying apoCys315Ala in the presence of FAD or 8-nor-8-chloroFAD, an analogue with an approximately 55 mV higher reduction potential. After correction for an estimated 65% reconstitutable apoprotein, the specific activity of apoCys315Ala in the presence of excess FAD or 8-nor-8-chloroFAD is 14% or 80%, respectively, of that observed with wild-type MSOX. Unlike oxidized flavin, apoCys315Ala exhibits a high affinity for reduced flavin, as judged by results obtained with reduced 5-deazaFAD (5-deazaFADH(2)) where the estimated binding stoichiometry is unaffected by dialysis. The Cys315Ala.5-deazaFADH(2) complex is also air-stable but is readily oxidized by sarcosine imine, a reaction accompanied by release of weakly bound oxidized 5-deazaFAD. The dramatic difference in the binding affinity of apoCys315Ala for oxidized and reduced flavin indicates that the protein environment must induce a sizable increase in the reduction potential of noncovalently bound flavin (DeltaE(m) approximately 120 mV). The covalent flavin linkage prevents loss of weakly bound oxidized FAD and also modulates the flavin reduction potential in conjunction with the protein environment.     

Purification and characterization of peroxisomal L-pipecolic acid oxidase from monkey liver

L-Pipecolic acid oxidase has been purified to near homogeneity from Rhesus monkey liver. The protein, a yellow monomer, has a molecular weight of 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.9. It contains a covalently bound flavin with absorption maxima at 457 and 383 nm and a shoulder at 480 nm. The purified enzyme is most reactive toward L-pipecolic acid, with lesser reactivities toward L-proline and sarcosine. The enzyme has no significant reactivity toward the D-enantiomer of pipecolic acid or toward any other amino acid tested. Benzoic acid is a competitive inhibitor of the enzyme with a Ki of 750 microM. The Km of the purified enzyme is 3.7 mM for L-pipecolic acid. With less purified preparations, the reaction product is alpha-aminodipic acid. The purified enzyme, however, produces an intermediate which reacts with ortho-aminobenzaldehyde to form an alpha-aminoadipic acid semialdehyde adduct. Thus, the formation of alpha-aminoadipic acid requires at least two enzymes.     

Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme.

BACKGROUND: Monomeric sarcosine oxidases (MSOXs) are among the simplest members of a recently recognized family of eukaryotic and prokaryotic enzymes that catalyze similar oxidative reactions with various secondary or tertiary amino acids and contain covalently bound flavins. Other members of this family include heterotetrameric sarcosine oxidase, N-methyltryptophan oxidase and pipecolate oxidase. Mammalian sarcosine dehydrogenase and dimethylglycine dehydrogenase may be more distantly related family members. RESULTS: The X-ray crystal structure of MSOX from Bacillus sp. B-0618, expressed in Escherichia coli, has been solved at 2.0 A resolution by multiwavelength anomalous dispersion (MAD) from crystals of the selenomethionine-substituted enzyme. Fourteen selenium sites, belonging to two MSOX molecules in the asymmetric unit, were used for MAD phasing and to define the local twofold symmetry axis for electron-density averaging. The structures of the native enzyme and of two enzyme-inhibitor complexes were also determined. CONCLUSIONS: MSOX is a two-domain protein with an overall topology most similar to that of D-amino acid oxidase, with which it shares 14% sequence identity. The flavin ring is located in a very basic environment, making contact with sidechains of arginine, lysine, histidine and the N-terminal end of a helix dipole. The flavin is covalently attached through an 8alpha-S-cysteinyl linkage to Cys315 of the catalytic domain. Covalent attachment is probably self-catalyzed through interactions with the positive sidechains and the helix dipole. Substrate binding is probably stabilized by hydrogen bonds between the substrate carboxylate and two basic sidechains, Arg52 and Lys348, located above the re face of the flavin ring.     

Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.

Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Structural characterization and mapping of the covalently linked FAD cofactor in choline oxidase from Arthrobacter globiformis

The flavoenzyme choline oxidase catalyzes the oxidation of choline and betaine aldehyde to betaine. Earlier studies have shown that the choline oxidase from Arthrobacter globiformis contains FAD covalently linked to a histidine residue. To identify the exact type of flavin binding, the FAD-carrying amino acid residue was released by acid hydrolysis. The fluorescence excitation maxima of the isolated aminoacylriboflavin, showing a hypsochromic shift of the near-ultraviolet band relative to riboflavin, and the pH-dependent flavin fluorescence confirmed the presence of an 8alpha-substituted flavin linked to histidine. Similarly, MALDI-TOF mass spectrometry showed a molecular mass corresponding to histidylriboflavin. Classical experiments used to distinguish between the N(1) and N(3) isomers all indicated that the flavin was linked to the N(1) position of the histidine residue. The position of the FAD-carrying histidine residue in the choline oxidase polypeptide was identified by tryptic cleavage of the denatured enzyme, HPLC separation of the proteolytic peptide fragments, and characterization of the purified flavin-carrying peptide by mass spectrometry and spectroscopy. The FAD moiety was assigned to the tryptic peptide, His-Ala-Arg, corresponding to residues 87-89 in the open reading frame of the previously published cDNA sequence. Further analysis of the flavopeptide by collision-induced dissociation mass spectrometry confirmed that the flavin cofactor was attached to His(87). We conclude that this variant of choline oxidase contains 8alpha-[N(1)-histidyl]FAD at position 87 in the polypeptide chain.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Covalent flavinylation of monomeric sarcosine oxidase: identification of a residue essential for holoenzyme biosynthesis

FAD in monomeric sarcosine oxidase (MSOX) is covalently linked to the protein by a thioether linkage between its 8alpha-methyl group and Cys315. Covalent flavinylation of apoMSOX has been shown to proceed via an autocatalytic reaction that requires only FAD and is blocked by a mutation of Cys315. His45 and Arg49 are located just above the si-face of the flavin ring, near the site of covalent attachment. His45Ala and His45Asn mutants contain covalently bound FAD and exhibit catalytic properties similar to wild-type MSOX. The results rule out a significant role for His45 in covalent flavinylation or sarcosine oxidation. In contrast, Arg49Ala and Arg49Gln mutants are isolated as catalytically inactive apoproteins. ApoArg49Ala forms a stable noncovalent complex with reduced 5-deazaFAD that exhibits properties similar to those observed for the corresponding complex with apoCys315Ala. The results show that elimination of a basic residue at position 49 blocks covalent flavinylation but does not prevent noncovalent flavin binding. The Arg49Lys mutant contains covalently bound FAD, but its flavin content is approximately 4-fold lower than wild-type MSOX. However, most of the apoprotein in the Arg49Lys preparation is reconstitutable with FAD in a reaction that exhibits kinetic parameters similar to those observed for flavinylation of wild-type apoMSOX. Although covalent flavinylation is scarcely affected, the specific activity of the Arg49Lys mutant is only 4% of that observed with wild-type MSOX. The results show that a basic residue at position 49 is essential for covalent flavinylation of MSOX and suggest that Arg49 also plays an important role in sarcosine oxidation.     

Abiotic Photophosphorylation Model Based on Abiogenic Flavin and Pteridine Pigments

A model for abiotic photophosphorylation of adenosine diphosphate by orthophosphate with the formation of adenosine triphosphate was studied. The model was based on the photochemical activity of the abiogenic conjugates of pigments with the polymeric material formed after thermolysis of amino acid mixtures. The pigments formed showed different fluorescence parameters depending on the composition of the mixture of amino acid precursors. Thermolysis of the mixture of glutamic acid, glycine, and lysine (8:3:1) resulted in a predominant formation of a pigment fraction which had the fluorescence maximum at 525 nm and the excitation band maxima at 260, 375, and 450 nm and was identified as flavin. When glycine in the initial mixture was replaced with alanine, a product formed whose fluorescence parameters were typical to pteridines (excitation maximum at 350 nm, emission maximum at 440 nm). When irradiated with the quasi-monochromatic light (over the range 325–525 nm), microspheres in which flavin pigments were prevailing showed a maximum photophosphorylating activity at 375 and 450 nm, and pteridine-containing chromoproteinoid microspheres were most active at 350 nm. The positions and the relative height of maxima in the action spectra correlate with those in the excitation spectra of the pigments, which point to the involvement of abiogenic flavins and pteridines in photophosphorylation.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.     

Purification and properties of glutamate synthase from Nocardia mediterranei.

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.     

Effects of chronic ethanol treatment on amino acid uptake and enzyme activities in the lactating rat mammary gland

The effects of chronic ethanol consumption on mammary gland amino acid uptake at the 15th day of lactation in the rat have been studied. Ethanol treatment decreased the arterial levels of Ala, Asp, Gly, Pro, Lys and Met, and increased those of Gln and alpha-amino-butyrate. Chronic ethanol treatment produced a decrease in the arteriovenous differences of Asp, Thr, Arg, Met and Phe, and increased those of Ala, Gln, Gly, Pro and Tyr. The combination of the calculated values of relative extraction and the arteriovenous differences indicate that these alterations in amino acid uptake are related to changes in the transport process for Ala, Asp, Thr, Pro, Arg, Asn, Gly, Tyr, and Phe, and that the alterations in the arteriovenous differences of Gln, Lys and Met are due to the affected arterial levels of these amino acids. Measurements of enzymatic activities in the mammary gland show that these alterations in the amino acid transport process cannot be ascribed to changes in the gamma-glutamyl cycle.     

Identification and properties of the covalently bound flavin of beta-cyclopiazonate oxidocyclase.

Beta-Cyclopiazonate oxidocyclase from Penicillium cyclopium has been previously shown to contain flavin dinucleotide in covalent linkage to the protein. In the present study, a pure flavin mononucleotide peptide was isolated from the enzyme by tryptic-chymotryptic digestion, chromatography on Florisil and on diethylaminoethylcellulose, and hydrolysis with nucleotide pyrophosphatase. The flavin peptide contains 9 amino acids, including histidine in linkage to the flavin, and Asx as the N-terminal residue. The fluorescence of the flavin in the FMN peptide is profoundly quenched even at pH 3.2, where protonation of the imidazole prevents queching of the flavin fluorescence by histidine. This quenching appears to be due to interaction of the flavin with a tryptophan residue, as the quenching is abolished by oxidation of the tryptophan with performic acid. Similarly, the fluorescence of the tryptophan in the peptide is quenched, presumably by the flavin. The flavin of beta-cyclopiazonate oxidocylcase is attached, by the way of the 8alpha-methylene group, to the imidazole ring of a histidine. The aminoacylflavin isolated from the enzyme is identical in the pKa of its imidazole group, in reduction by NaBH4, and in other properties with synthetic 8alpha-(N1-histidyl)riboflavin. The pKa of the histidylriboflavin component of the oxidocyclase is 5.2 before and 5.0 after acid modification of the ribityl chain, as is found in the synthetic derivative. It is concluded that the enzyme contains the N1 isomer of histidylriboflavin and that acid hydrolysis of flavin peptides isolated from the oxidocyclase, while liberating histidylriboflavin, also causes acid modification of the ribityl chain of the flavin moiety.     

On the presence of a novel covalently bound oxidation-reduction cofactor,iron and labile sulfur in trimethylamine dehydrogenase

Trimethylamine dehydrogenase from a facultative methylotroph contains 4 g atoms each of Fe and S and an unknown, covalently bound, yellow coenzyme. The absorbance of the enzyme in the visible range (λmax=445 nm) is extensively bleached by dithionite. Reduction by substrate causes less extensive bleaching and the appearance of a three banded spectrum which may be representative of a free radical form. Denaturation liberates the FeS center(s) but not the organic coenzyme. The latter is covalently linked to the protein via an amino acid residue and is solubilized on proteolytic digestion in the form of the peptide. The coenzyme-peptide has been purified to a constant ratio of amino acid to coenzyme. The oxidized and reduced forms show maximal absorbance at 437 nm and 380 nm respectively. Based on dithionite titrations its molar absorbance at 437 nm is 12,300 in the oxidized and 4000 in the dithionite reduced form. The cofactor is very labile to photolysis giving rise to several products the predominant one of which shows fluorescence excitation and emission maxima at 394 and 500 nm, respectively. After cleavage of the hydrolyzable amino acids in HCl, the compound consumed 3 moles of periodate. Digestion with aminopeptidase M yields a compound with a single amino acid and ～1 mole of organic P present. Acid phosphatase, but not nucleotide pyrophosphatase affects its mobility. These findings suggest that the coenzyme-peptide is isolated in the form of a mononucleotide, containing a 5-carbon alcohol. The physical and chemical properties of the compound do not agree with those of known flavin or pyridoxine derivatives but are not incompatible with a covalently linked pteridine (lumazine) derivative, although no proof for such a structure is so far available.     

Role of Glu318 at the putative distal site in the catalytic function of cytochrome P450d.

Most microsomal P450s have a conserved "threonine cluster" composed of three Thrs (Thr319, Thr321, Thr322 for P450d) at a putative distal site. An ionic amino acid at 318 is also well conserved as Glu or Asp for most P450s. To understand the role of these conserved polar amino acids at the putative distal site in the catalytic function of microsomal P450, we studied how mutations at this site of P450d influence the activation of molecular oxygen in the reconstituted system. Catalytic activity (0.02 min-1) toward 7-ethoxycoumarin of the Glu318Ala mutant of P450d was just 6% of that (0.33 min-1) of the wild type, while those of Glu318Asp, Thr319Ala, and Thr322Ala were comparable to or even higher than that of the wild type. Consumption rates of O2 and formation rates of H2O2 of those mutants varied in accord with the catalytic activities. Especially, the efficiency (0.5%) of incorporated oxygen atom to the substrate versus produced H2O2 for the Glu318Ala mutant was much lower than that (3.7%) of the wild type, while that (58.8%) for the mutant Glu318Asp was 16-fold higher than that of the wild type. In addition, the autoxidation [Fe(II)---- Fe(III)] rate (0.074 s-1) of the Glu318Ala mutant was much lower than those (0.374-0.803 s-1) of the wild type and other mutants. Thus, we strongly suggest that Glu318 plays an important role in the catalytic function toward 7-ethoxycoumarin of microsomal P450d.