Metal Cofactor Specifics

TABLE 7.5 Bond Distances in Cytochrome b(6)f Hemes for PDB 1VF5 

Heme Complex Subunit/Heme-Protein Connection Complex Subunit/Fe Ligands (in Addition to Porphyrin N Atoms) Bond Distance (A) 

Heme f Cyto f subunit/Covalent Cyto f subunit  

Rieske [2Fe-2S] (S -C = 1.83 A) and cys25 (Sy-C = 1.84 A) to carbon atoms of the porphyrin s C=CH substituents tyrl backbone amide N Rieske iron-sulfur protein (RISP or ISP) cysl08 Sy-Fei cysl26 Sy-Fei hisllO Nsi-Fe2 hisl29 Nsi-Fe2 2.34 2.32 2.32 2.27 2.25 

Heme x Cyto bg subunit helix A cys35 Sy bonded to carbon atom of the porphyrin s C=CH substituent (Sy-C = 1.78 A) HjO-Fe Fe axial H2O ligand hydrogen-bonded to propionate side chain of heme bn (0H2---0 = 3.50 A) and cyto be gly38 backbone amide N-H (OH2-N-H = 2.70 A) 2.53 

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Cleavage in the presence of histidine releases the oligonucleotide fragment containing the random sequence, and this is amplified by PCR and the cycle repeated. This selection procedure produced deoxyribozymes that require no metal cofactor but have a specific requirement for L-histidine, which is presumed (from pH-rate profiles) to act as a general... 

In view of these problems with Ni2+, Mna+ has been used as a probe for Mg2+ with some success. However, it should be noted that there is a difference in radius which may be manifested in different biochemical behaviour (Mn2+, 0.80 A Mg2+, 0.65 A). Thus Mn2+ has been used to probe the Mg2+ site in pyruvate kinase.95 While the Mg2+-activated enzyme is inhibited by Ca2+ and Li+, the Mn2+-activated enzyme is inhibited by Ca2+ and not by Li+. There are also differences in the catalytic and regulatory properties of the NAD+-specific malic enzyme of E. coli,104 depending upon whether the divalent activator is Mn2+ or Mg2+. It is necessary, therefore, to express a cautionary note. These two cations may act in slightly different ways to bring about a similar final result. In the second example it appears that the metal cofactors stabilize two different conformational states of the enzyme. 

All proteolytic enzymes described are fairly non-specific serine endoproteases, cleaving peptide chains preferentially at the carboxyl side of hydrophobic amino acid residues. The enzymes convert their substrates into small, readily soluble fragments which can be removed easily from fabrics. Only serine protease can be used in detergent formulations, as thiol proteases such as papain would be oxidized by the bleaching agents, acidic proteases are not active at common laundry conditions, and metalloproteases such as thermolysin would lose their metal cofactors because of complexation with the water-softening agents or hydroxyl ions. 

Customize RDF File. . . Add specific metals, cofactors, and/or water molecules or determine protonation statpf ASP, CYS, GLU, HIS, SER... 

Fig. 2. Creation of the receptor description file (RDF) for use with FlexX. Protein Structure Specify the correct PDB structure. Active-Site File The FlexX binding pocket can be defined as the amino acid residues within 7 A from a reference structure (e.g., a ligand structure translated to the protein active site midpoint as determined by PASS see Subheading 3.3.2.). Customize RDF File enables specification of metals, cofactors, protonation states, and torsions of residues (see Fig. 3).

Fig. 3. Customization of the receptor description file. Chains allows specification of the chains present in the protein that have to be included (grayed out if only one chain is present) Templates to add specific metals, cofactors, and/or water molecules or determine protonation state of Asp, Cys, Glu, His, Ser Torsions allows setting of the dihedral angle of the terminal hydrogen atom of the specified residue.

The simplest use of manganese is as part of a metal cofactor such as manganese(II) adenosine triphosphate, [Mn(II)ATP2-]. The metal complex is usually found as the bidentate [Mn(II)0,7-ATP2 ] (3, 4) as illustrated in Figure 1. Enzyme specificity for Mg(II), Mn(II), or Ca(II) ATP complexes is dependent on a variety of factors however, once selected, each metal apparently functions in an analogous manner. The metal ion serves two main purposes. First, based on the coordination geometry... 

Proline hydroxylase has been isolated and characterized and is known to contain a mononuclear nonheme ferrous iron center that is the catalytic active site of the enzyme, coordinated by two histidine and one aspartate side chains. The requirement for Fe(II) is reflected in the in vivo sensitivity of collagen formation to chelators specific for ferrous ion (e g. 2,2 -dipyridyl). In addition to the catalytic metal cofactor, the reaction requires a reducing cosubstrate, 2-oxoglutarate, dioxygen, and the procollagen peptide (equation 1). 

The D-mannanase produced by Bacillus subtilis83 was found to show a dependency towards Ca2+ for the maintenance of activity and stability. In the presence of Ca2+, this enzyme preparation showed optimal activity at pH 6.0 and 60°, and was stable at pH values of 5.0-9.5. The enzyme was completely inactivated when kept at 70° for 15 min at pH 7.5. In the presence of EDTA, however, the enzyme showed optimal activity at pH 6.0 and 40°, was stable only at pH values of 6.0 to 7.5, and was completely inactivated at 55°. Temperature and pH properties of D-mannanases (from other sources) not dependent on metal cofactors are summarized in Table XII, together with the pi and M.W. values for several D-mannanases. These enzymes are considered to be acid glycan hydrolases, and their sizes (M.W. range, 22-47 X 104) are comparable to those of other polysaccharases (see, for example, Sections II, III, and VI). Other physical properties of D-mannanases that have been measured include the partial specific volume (0.72 cm3.g 1, for the Aspergillus niger84 enzyme) and the sedimentation coefficient (3.85, for the A. niger84 enzyme). 

These chelating resins have found most of their use in metal ion recovery processes in the chemical and waste recovery industries. They may find use in fermentation applications where the cultured organism requires the use of metal ion cofactors. Specific ion exchange resins have also been used in laboratory applications that may find eventual use in biotechnology product recovery applications. 

Unlike S-COMT from rat liver, COMT (alfalfa) is also presumed to utilize a general base without the aid of any metal cofactor for catalysis. Specifically, methyl transfer from SAM (115) to caffeic acid (5) and/or... 

Dong s group reports a simple, sensitive, and label-free I7E DNAzyme-based sensor for Pb + detection. The catalytic activity of some DNAzymes is divalent metal ion-specific, just as the catalytic activity of some protein enzymes is metal ion cofactor-dependent (Santoro and Joyce, 1997 Carmi et al., 1998). In this work, the authors focus on 17E DNAzyme, which is a divalent Pb -specific enzyme employed widely in Pb + sensors (Santoro and Joyce, 1997). As shown in Figure 12.16A, in the presence of Pb, 17E DNAzyme could cleave the substrate 17DS, which could release ssDNA (including 17E and fragments from 17DS)... 

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