Half-cystinyl Residues Research Articles

Effect of sulfhydryl reagents on phenolase action

The conversion of [4- 14C] estradiol to its 2-hydroxy derivative by mushroom phenolase in the presence of NADH was inhibited by N-ethylmaleimide and p-chloromercuribenzoate without affecting the formation of any of the other metabolites. It is proposed that these sulfhydryl reagents exert their effect by combining with the half-cystinyl residues of the enzyme reduced by NADH.

3,4-Dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione 4,5-Dioxygenase from Nocardia restrictus: I. ISOLATION OF THE ENZYME AND STUDY OF ITS PHYSICAL AND CHEMICAL PROPERTIES

Abstract 3,4-Dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione-4,5-dioxygenase was purified to a state of apparent homogeneity from Nocardia restrictus. The molecular weight of the enzyme was estimated to be around 280,000 from sedimentation equilibrium and sedimentation velocitydiffusion data. The physicochemical properties described include: sedimentation coefficient (s020,w = 10.42 S), diffusion coefficient (D020,w = 2.55 x 10-7 cm2 sec-1), frictional ratio (f/f0 = 1.47), and apparent partial specific volume (v = 0.733 ml g-1). The enzyme has an ultraviolet absorption maximum at 280 mµ, and the specific absorbance (E0.1%1 cm) is 0.93. Quantitative analytical data show 1.13 g-atoms of ferrous iron and 9 half-cystinyl residues per mole of the enzyme. Metal-chelating agents, including o-phenanthroline, 8-hydroxyquinoline, and α,α'-dipyridyl, inhibited the enzyme, and the inhibition was noncompetitive with respect to both organic substrate and molecular oxygen. Sulfhydryl inhibitors also inhibited the enzyme at the concentration of 1 mm. Substrate specificity studies indicated that substitution of an alkyl group at a position adjacent to the dihydroxyl group was required for maximal activity. Introduction of a bulky alkyl group at other positions rendered the substrate less reactive.

The Disulfide Bonds of Bovine α-Lactalbumin

The four disulfide bonds formed by the 8 half-cystinyl residues in α-lactalbumin have been established by sequence analysis of peptides isolated from enzymic digests. Two unique disulfide-containing peptides were isolated by chromatography from peptic digests of α-lactalbumin. A third peptic peptide contained 4 half-cystinyl residues. Further degradation of this peptide with thermolysin gave two additional, unique disulfide-containing peptides that were readily separated by column chromatography. Partial sequence analysis of each of the four unique disulfide peptides after reduction and carboxymethylation provided an unequivocal assignment of the four disulfide bonds in α-lactalbumin. The four disulfide bonds are formed through linkages between Residues 6 and 120, Residues 28 and 111, Residues 61 and 77, and Residues 73 and 91. This arrangement of the disulfide bonds is essentially identical with that found in chicken egg-white lysozyme.

The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes.

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.

β-Hydroxydecanoyl Thioester Dehydrase: STUDIES ON MOLECULAR STRUCTURE AND ACTIVE SITE

Abstract β-Hydroxydecanoyl thioester dehydrase from E. coli has been shown to have a molecular weight of 36,000 by ultracentrifugation, gel filtration, and amino acid composition. Electrophoretic behavior, however, suggests that the protein molecule is composed of two equal, if not identical, polypeptide chains of molecular weight 18,000. The enzyme also contains 4 half-cystinyl residues per 36,000 g of protein. A variety of chemical modifications, including photooxidation, alkylation, and nitration, lead to the conclusion that 1 histidyl and 1 tyrosyl residue are essential for the catalytic activity of the enzyme. The pH-kcat profile for the isomerization reaction is consistent with the participation of these two amino acids in the catalytic process. Evidence is presented that 3-decynoyl-N-acetylcysteamine, a specific dehydrase inhibitor, reacts covalently with the same histidyl residue and protects the enzyme against the modifying active site-directed reagents. Conversely, nitrated or alkylated enzyme has lost the ability to react with the acetylenic inhibitor. No treatment has been found which will dissociate dehydration from isomerization activity, supporting the concept that the dehydrase is a truly multifunctional enzyme.

The amino acid sequence of bovine carboxypeptidase A.

The amino acid sequence of the four fragments produced by treatment of bovine carboxypeptidase A with cyanogen bromide has been completed. The alignment of these fragments, previously established by peptic digest of the whole protein, allows for the description of the complete primary structure of the molecule. A comparison of the proposed functional residues, identified by X-ray diffraction analyses, has either confirmed their assignments or provided their correct identity. The functional and structural residues of principal importance include Arg 145, Tyr 248, and Glu 270 as the binding site of the substrate carboxyl group, the proton donor, and the nucleophilic moiety, respectively, which were correctly assigned; His 196 as the third zinc ligand and Tyr 265 as the binding site of the alpha-carboxyl group have been corrected from their original X-ray assignments. The other two zinc ligands, His 69 and Glu 72, were identified previously from chemical and X-ray studies. The assignment of the two half-cystinyl residues and the postulation of the existence of a disulfide bond have been confirmed.

The amino acid sequence of cobrotoxin

Reduced S-carboxymethylated cobrotoxin was digested with trypsin and chymotrypsin. Nine peptides were isolated from the tryptic digest by a combination of electrophoresis and chromatography on paper. The amino acid sequence in these peptides was determined by applying the Edman degradation and by using proteolytic enzymes, leucine aminopeptidase and carboxypeptidases. The arrangement of nine peptides in a linear structure was determined by comparing the amino acid compositions of the overlapping peptides isolated from the chymotryptic digest of reduced S-carboxymethylated cobrotoxin. The striking distributional regularities of the basic and hydrophilic residues in the sequence and the importance of the positions of half cystinyl residues which form the disulfide bonds for maintaining the protein in its active configuration, are considered in connection with the structure-activity relationship of the protein.

REDUCTION AND REOXIDATION OF THE DISULPHIDE BONDS OF PEPSINOGEN.

Anfinsen and Haber have shown that the disulphide bonds of ribonuclease (RNase) can be cleaved by treatment of the native protein in 8 M urea with mercaptoethanol1. On exposure to molecular oxygen, reduced RNase is re-oxidized with complete regeneration of secondary and tertiary structure. The kinetics of the reaction indicate either that pairing of the correct half-cystine residues is a random process with the molecule undergoing subsequent structural rearrangement to yield the native form2; or, alternatively, that the reduced protein may be sufficiently structurally similar to the native enzyme to allow correct matching and reoxidation of the half-cystinyl residues, even in the absence of the disulphide bridges3.