Copper Polypeptides

One specific question which I would like to address to Dr. Margerum concerns the very interesting series of substitution reactions for the polypeptide complexes that are of similar stability and yet exhibit remarkably different substitution rates. What would you say about the implications of that observation for the formation processes of the copper polypeptide complexes ... 

These findings suggest that the copper transported through the intestinal membrane is bound to polypeptides. The exact structure of these polypeptides is not clear, but, as is mentioned later, copper polypeptides have been found in the urine of patients with Wilson s disease. The relation between intestinal absorption and urinary excretion remains to be investigated. 

Uzman and his associate [55] proposed that the disease results from a general disturbance in protein metabolism (possibly increased proteolytic activity) leading to the appearance in tissues of substances of great binding affinity for copper. The evidence in favor of this concept is twofold first, copper peptides were found in the urine of patients with Wilson s disease and, second, electrophoretic studies of liver proteins revealed the presence of abnormal copper proteins. This interesting hypothesis merits further confirmation. It will also be necessary to explain why abnormal copper polypeptides lead to lower ceruloplasmin levels. 

Figure 7. Traces of the a-carbon polypeptide backbone of domains 1 and 6 in the hCP structure. Domain 1 is shown (shaded) on the left hand side of the diagram this domain contributes four histidine residues (not shown) to the trinuclear cluster copper atoms are depicted as black spheres. Domain 6 is on the right hand side of the figure and also contributes four histidine residues to the cluster. The portion of the polypeptide chain colored black is that which is missing in the truncated enzyme. This polypeptide, residues 991 to 1046 inclusive, includes two histidine residues bound to the trinuclear copper center and three residues bound to the mononuclear copper in domain 6 these residues are depicted in black. The absence of the C-terminal polypeptide would also remove over 50% of the interdomain hydrogen-bond and iron-pair interactions observed in the intact enzyme.

Metallothionein was first discovered in 1957 as a cadmium-binding cysteine-rich protein (481). Since then the metallothionein proteins (MTs) have become a superfamily characterized as low molecular weight (6-7 kDa) and cysteine rich (20 residues) polypeptides. Mammalian MTs can be divided into three subgroups, MT-I, MT-II, and MT-III (482, 483, 491). The biological functions of MTs include the sequestration and dispersal of metal ions, primarily in zinc and copper homeostasis, and regulation of the biosynthesis and activity of zinc metalloproteins. 

Copper enzymes are involved in reactions with a large number of other, mostly inorganic substrates. In addition to its role in oxygen and superoxide activation described above, copper is also involved in enzymes that activate methane, nitrite and nitrous oxide. The structure of particulate methane mono-oxygenase from the methanotrophic bacteria Methylococcus capsulatus has been determined at a resolution of 2.8 A. It is a trimer with an a3P33 polypeptide arrangement. Two metal centres, modelled as mononuclear and dinuclear copper, are located in the soluble part of each P-subunit, which resembles CcOx subunit II. A third metal centre, occupied by Zn in the crystal, is located within the membrane. 

Mammalian COX (the illustration shows the enzyme from bovine heart) is a dimer that has two identical subunits with masses of 204 kDa each. Only one subunit is shown in detail here the other is indicated by gray lines. Each subunit consists of 13 different polypeptides, which all span the inner mitochondrial membrane. Only polypeptides I (light blue) and II (dark blue) and the linked cofactors are involved in electron transport. The other chains, which are differently expressed in the different organs, probably have regulatory functions. The two heme groups, heme a (orange) and heme ai (red) are bound in polypeptide 1. The copper center Cua consists of two copper ions (green), which are coordinated by amino acid residues in polypeptide II. The second copper (Cub) is located in polypeptide I near heme... 

Fig. 2. Copper site in azurin. In this and subsequent figures the following conventions have been used, (a) The copper site is generally an enlargement of (b). The copper site is a dotted sphere, the ligand residues are represented by bonds connecting atoms in the side chain, and, where possible, the atoms bonded to the copper atom are identified by atom type. Ribbons represent portions of the backbone structure near the copper. In (b) of each figure, the main-chain polypeptide is represented by a ribbon fit to the main-chain coordinates, and the amino and carboxy termini are indicated by N and C, respectively, (c) A schematic version drawn from (b). Solid arrows represent main-chain regions participating in the /3 sheet roughly above the plane of the paper, while dotted or light arrows are the...

A new putative member of the blue multi-copper oxidases has been isolated using the Escherichia coli yacK gene. Six copper ions per polypeptide chain were determined and assigned to two type 1 copper centers and further one type 2 and one type 3 copper. Phenoloxidase and ferroxidase properties were ascertained98 A new copper containing nitrite reductase was purified from a halophilic archaeon and the ligands to type 1 and type 2 coppers in the sequence were... 

The blue oxidases contain these three types of copper together The stoichiometry is straightforward with laccase which contains one type-1 and one type-2 copper, and one type-3 dimeric copper site . One would expect two laccase-like sites in ascorbate oxidase and in ceruloplasmin, but the presence of respectively 3 and 1 and 1 and 3 type-1 and type-2 copper atoms has been deduced. Ceruloplasmin shows oxidase activities towards different substrates, like Fe (ferroxidase) and aromatic amines. It plays, moreover, an active role in the transport of copper With the proper precautions against the action of proteinases it can be isolated as a single polypeptide chain... 

Less sensitive but very simple and precise is measurement of the light absorption around 280 nm. This is discussed in the main text in Section D,6. For a typical protein an absorbance of 1.0 at 280 nm corresponds to a protein concentration of 1 mg / ml.h The very old biuret method is also useful for samples containing 1-10 mg / ml of protein. The violet color that arises upon addition of copper sulfate to an alkaline solution of a peptide or protein is recorded at 540-560 nm.h The color is especially intense for longer polypeptides. The name of the method arises from the fact that biuret gives a similar color1 (see also Eq. 6-85). 

The single 639-residue polypeptide chain contains one type 2 copper ion.554 Neither oxygen nor galactose affects the absorption spectrum of the light murky green enzyme, but the combination of the two does, suggesting that both substrates bind to the enzyme before a reaction takes place. A side reaction releases... 

Starting at the far left, we see a water molecule, two common amino acids, alanine and tryptophan, a segment of a DNA double helix, a segment of a protein single helix, and the folded polypeptide chain of the enzyme copper, zinc superoxide dismutase or SOD. 

Plastocyanin consists of a single polypeptide chain of molecular weight around 10 500 and one copper atom. It is synthesized in the cytoplasm as a precursor of higher molecular weight,911 with an additional polypeptide that is essential for transport of the protein into the chloroplast. Within... 

The azurins are electron-transfer proteins in the respiratory chains of certain bacteria. They have been particularly well studied from Pseudomonas aeruginosa and other pseudomonads, and contain one type 1 copper bound to a single polypeptide chain of molecular weight about 16 000. Amino acid sequence data for a number of azurins show that about one third of residues are conserved. All contain three cysteine residues. Three are also sequence homologies with the plastocyanins. 

There has been much uncertainty over the molecular weight and copper content. It appears to involve a single polypeptide chain of molecular weight about 130 000 with six or seven copper ions. On balance, there are two type 1, one type 2 and four type 3 copper centres. This is in accord with the fact that 44% of the total copper is paramagnetic and ESR-detectable.977... 

The cytochrome o from Azotobacter vinelandii is reported to consist of one polypeptide of molecular weight 28 000 with two identical heme components. It has also been isolated from the thermophile PS3,1319 Escherichia coli, Vitreoscilla, Pseudomonas aeruginosa, and Rhodopseudomonas spp. The enzyme from Vitreoscilla consists of two identical polypeptides of molecular weight 13 000 and two moles of protoheme IX. A cytochrome b562-o complex from E. coli contains two peptides and, strangely, copper.1320... 

Ceruloplasmin is the major copper-containing protein in mammalian blood plasma (see Section 62.1.8.5), and appears to consist of a single polypeptide chain of molecular weight 130 000.1341 The nine-line superhyperfine splitting in the ESR spectrum of the type 2 copper has been interpreted in terms of four equivalent nitrogen ligands.978... 

Metal-binding polypeptides in roots of copper-tolerant Mimulus... 

U Tessmer, R Dernick. Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera. Electrophoresis 10 277-279, 1989. 

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