Quantitation of aromatic residues in proteins: model compounds for second-derivative spectroscopy

Levine, Rodney L.; Federici, Marcia

doi:10.1021/bi00540a004

Cited by 129 publications

(69 citation statements)

References 38 publications

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“…Such analysis allows greater ability to distinguish individual spectra in a composite spectrum, which otherwise might be difficult to resolve. Similar analyses have been described for the three aromatic amino acids [37].…”

supporting

confidence: 55%

The auto‐oxidation of tetrahydrobiopterin

Davis

Kaufman

Milstien

1988

European Journal of Biochemistry

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The product of the aerobic oxidation of tetrahydrobiopterin, quinonoid dihydrobiopterin, is unstable and rapidly rearranges to form a 7,s-dihydropteridine. Kaufman [Kaufman, S. (1967) J. Biol. Chem. 242,3934-39431 identified the stable product produced in 0.1 M phosphate pH 6.8, as 7,s-dihydrobiopterin. However, Armarego et al. [Armarego, W. L. F., Randles, D. and Taguchi, H. (1983) Eur. J. Biochem. 135 393-4031 questioned this assignment because they found that the dihydroxypropyl group on C-6 was eliminated and 7,s-dihydropterin was the predominant product when the aerobic oxidation was performed in 0.1 M Tris pH 7.6. In the present study we demonstrate that the rearrangement of the unstable quinonoid dihydrobiopterin results in a mixture of these two 7,8-dihydropteridines at neutral pH, 25 "C. Furthermore, we find that the loss or retention of the alkyl sidechain is not solely dependent on the pH of the reaction mixture, as was previously assumed by Armarego et al., but rather is strongly influenced by the temperature and the type of buffer. In addition, we describe a new method for quantifying the relative amounts of these two 7,8-dihydropteridines in mixtures of unknown concentrations. This method relies on multicomponent analysis of second derivative spectra and results in values which agree with the concentrations determined directly by HPLC.A reduced pteridine cofactor is necessary for phenylalanine hydroxylase to catalyze the conversion of phenylalanine to tyrosine [l]. The natural cofactor for this enzyme (as well as for tyrosine hydroxylase [2-51 and tryptophan hydroxylase [6, 71 is tetrahydrobiopterin, (BH,) [S]. During the hydroxylation reaction at neutral pH, there is a stoichiometric oxidation of BH4 to quinonoid dihydrobiopterin (qBH2) [9, 101. This latter species can then be reduced back to BH4 by the NADH-dependent enzyme, dihydropteridine reductase [ l l -151. qBH2 is unstable and, in the absence of the reductase, rapidly rearranges to a 7,s-dihydropteridine [9]. It was concluded that 7,s-dihydrobiopterin (BH,) was the product of the tautomerization of qBH2 [16,17] because when the auto-oxidation of BH4 was performed in 0.1 M phosphate pH 6.8 at ambient temperature, the ultraviolet spectrum of the product was identical to that of authentic BH2 [16]. This conclusion was also strongly supported by the finding that one of the products of the aerobic oxidation of BH4 in phosphate buffer was a substrate for sepiapterin reductase, an enzyme that catalyzes the NADP-dependent oxidation of the 2'-hydroxyl group on the side-chain of BH2 [16].However, it has become apparent that the unstable qBH2 does not always isomerize to BH,. Whereas the oxidation of Correspondence to

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supporting

confidence: 55%

The auto‐oxidation of tetrahydrobiopterin

Davis

Kaufman

Milstien

1988

European Journal of Biochemistry

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“…Aromatic amino acid contents were determined by absorption spectroscopy of enzyme denatured in 6 M guanidinium chloride [13].…”

Section: Methodsmentioning

confidence: 99%

Methylamine oxidase from Arthrobacter P1. A bacterial copper-quinoprotein amine oxidase

1986

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Methylamine oxidase from Arthrobacter P1 was purified to homogeneity. The enzyme oxidizes primary amines but not tyramine or polyamines like spermine and putrescine. The enzyme activity has a pH optimum of 8.0 with methylamine, and is inhibited by certain cations as well as anions at rather low concentrations. The enzyme has an M , of 167900, an isoelectric point of 4.6, consists of two (probably identical) subunits (Mr 82250) and contains two copper atoms but no sugar residues. The visible absorption spectra of the enzyme as it is isolated (broad maximum at 480 nm), that of its reduced form obtained on addition of excess of methylamine (maximum at 470 nm) and that of phenylhydrazine-inhibited enzyme (maximum at 440 nm) are very similar to those of eucaryotic copper-containing amine oxidases (EC 1.4.3.6). Also the stoichiometry of inhibition with carbonyl group reagents is similar since the enzyme reacted with only one methylhydrazine. The adduct isolated from copper-free enzyme, treated with 2,4-dinitrophenylhydrazine, was identical to that found in bovine serum amine oxidase treated with t h s compound after copper removal. This indicates that the enzyme is a copper-quinoprotein amine oxidase, the first example from bacterial origin.A large number of methylotrophic bacteria are able to convert methylamine directly into formaldehyde and ammonia [I]. Within this group, all gram-negative bacteria studied so far use NAD(P)-independent, periplasma-located methylamine dehydrogenase (EC 1.4.99.3) for that purpose. [5], and probably also from yeasts [6], are copper-containing enzymes. In this light it was interesting to purify methylamine oxidase from Arthrobacter P1 and to look for the presence of flavin and copper.During these studies, it was found that this enzyme had all the characteristics of a copper-containing amine oxidase. Since such an enzyme also contains an organic prosthetic group, the nature of which was recently established to be pyrroloquinoline quinone (PQQ) in bovine serum amine oxidase [7], attempts were made to provide evidence for the existence of this compound in methylamine oxidase. MATERIALS AND METHODS Cell culturingArthrobacter P1 (kindly provided by Prof. W. Harder) was cultured aerobically at 30°C on a mineral medium 121 with 0.5% methylamine . HCl as a carbon and energy source. The cells were harvested in the stationary phase, washed with 50 mM potassium phosphate, pH 7.0, and stored at -20°C. Enzyme purificationThawed cells (84 g wet weight) were suspended in 125 ml 50 mM potassium phosphate, pH 7.0, and treated during 5 min by an ultrasonic disintegrator (MSE, 150 W) with cooling. After centrifugation, the pellet was resuspended in 100 ml buffer and the sonification repeated for 10 min. The supernatants were combined (225 ml) and 24.3 g poly(ethy1-ene glycol) (PEG) 6000 was added (10% w/v) with stirring for 20 min at room temperature. After centrifugation, further protein precipitation was achieved by adding 155.7 g PEG 6000 to the supernatant (50% w/v) and stirring for 90 min. Precipi...

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“…The remainder of the sample was then dialyzed at 4º, using dialysis cassettes (Pierce, Rockford, IL), against 5 mM HEPES, 10 mM sodium chloride at pH 7.4, with six changes of buffer, and then dried by vacuum centrifugation (Savant). To measure tryptophan by spectral deconvolution (26), 350 :g of "1-AT were used; following reaction, "1-AT was dialyzed against 5 mM potassium phosphate, pH 5.0, with six changes of buffer.…”

mentioning

confidence: 99%