Histidine residues replacement

Early mutational studies of the Rieske protein from 6ci complexes have been performed with the intention of identifying the ligands of the Rieske cluster. These studies have shown that the four conserved cysteine residues as well as the two conserved histidine residues are essential for the insertion of the [2Fe-2S] cluster (44, 45). Small amounts of a Rieske cluster with altered properties were obtained in Rhodobacter capsulatus when the second cysteine in the cluster binding loop II (Cys 155, corresponding to Cys 160 in the bovine ISF) was replaced by serine (45). The fact that all four cysteine residues are essential in Rieske clusters from be complexes, but that only two cysteines are conserved in Rieske-type clusters, led to the suggestion that the Rieske protein may contain a disulfide bridge the disulfide bridge was finally shown to exist in the X-ray structure (9). 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

The large molecule consists of a single peptide chain 35% P-sheet and 20% helical structure are found in the folded stmcture. The active site is a 1.2 nm deep conical cavity in the central pleated sheet, with a ion located at its bottom. Three histidine residues hold the Zn +, which also binds an HjO molecule. The active-site cavity is divided into hydrophilic and hydrophobic halves. The inhibitors of the enzyme replace the water on the Zn + ion and also block the fifth coordination site where COj should bind. 

Bovine pancreatic RNase A is a member of a homologous superfamily. In addition, there is a separate family of guanine-specific microbial RNases that have evolved to have a similar active site.192,193 Ribonuclease T1 from Aspergillus oryzae and the 110-residue bamase from Bacillus amyloliquefaciens of Mr 12 392 (see Chapter 19) are the best known examples. One of the histidine residues is replaced by a glutamate in these enzymes. The microbial enzymes are much more amenable to study by protein engineering. 

Yi X, Mroczko M, Manoj KM, Wang X, Hager LP (1999) Replacement of the Proximal Heme Thiolate Ligand in Chloroperoxidase with a Histidine Residue. Proc Natl Acad Sci U S A 96 12412... 

Peptide Chloromethyl Ketones. Peptide chloromethyl ketone inhibitors have been studied extensively and a fairly detailed picture of the inhibition reaction (see Figure 3) has emerged from numerous chemical and crystallographic studies (30,31). The inhibitor resembles a serine protease substrate with the exception that the scissile peptide bond of the substrate is replaced with a chloromethyl ketone functional group in the inhibitor. The inhibitor binds to the serine protease in the extended substrate binding site and the reactive chloromethyl ketone functional group is placed then in the proper position to alkylate the active-site histidine residue. In addition, the serine OH reacts with the inhibitor carbonyl group to form a hemiketal. 

In normal Hb, the v(Fe—N) of the proximal histidine (F8) is near 220 cm-1 (6). In mutant Hb such as Hb M Iwate and Hb M Boston, F8 histidine and E7 histidine are replaced by tyrosine residues, respectively. In five-coordinate ferric a subunits of these compounds, the v(Fe—O- (phenolate)) bands are observed at 589 and 603 cm-1, respectively (7). 

An important structural difference between eliminases and mutases concerns the axial base coordinated to the cobalt, perpendicular to the plane of the corrin ring. Whereas in all the eliminases, the axial base is the dimethylbenzimidazole of the coenzyme itself (Fig. 1), the mutases use a conserved histidine residue of the enzyme for this purpose. On binding of the coenzyme to the apo-enzyme, the axial base is replaced by the histidine and moves into a distinct pocket of the protein. Methyltransferases (see below) also use a protein histidine as axial base, whose reactivity is fine tuned by protonation. Possibly the mutases and methyltransferases have a common evolutionary origin, whereas the eliminases evolved separately. 

Base-off, his-on means that the 5,6-dimethylbenzimidazole ligand (cf. Fig. 1) is detached and replaced by a histidine residue from the protein partner base-on means that the 5,6-dimethylbenzimidazole ligand is still ligated to cobalt. 

In the expression of granulocyte colony stimulating factor (G-CSF), Lu and coworkers have characterized a mistranslation product where a histidine residue has been replaced by a glutamine (5). Elimination of such problems is important to the biotechnology industry it is thus essential to imderstand the biological mechanisms that are involved. 

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