Native enzymes

The values of the Michaelis-Menten kinetic parameters, Vj3 and C,PP characterise the kinetic expression for the micro-environment within the porous structure. Kinetic analyses of the immobilised lipase in the membrane reactor were performed because the kinetic parameters cannot be assumed to be the same values as for die native enzymes. 

All these immobilization techniques run the risk of altering activity compared with the native enzyme. Improved activity is occasionally reported, but this is the exception. The immobilization techniques listed above are in approximate order of loss in activity. Physical entrapment normally causes no change. Adsorption will distort the shape of the molecule compared with the native... 

Ni, but it had the native enzyme oligomeric organization (46). Thus, the C-terminal extension is needed for Ni incorporation, which, in turn, is not needed for subunit assembly. 

The two subunits of CODH/ACS have been dissociated to offer a clearer picture of the ACS active site 135). The holoenzyme contains 2 Ni, 12 Fe, and 14 S 120) that are organized into 3 discrete clusters, whereas the isolated a subunit contains only 1 Ni and 4 Fe and has spectroscopic properties similar to those of Cluster A in the native enzyme 186). Based on EXAFS spectroscopy of the a. subunit, the Ni site in Cluster A has been proposed to be coordinated to 2 sulfur ligands at 2.19 A and 2 nitrogen or oxygen ligands at 1.89 A in a distorted square plane 186). 

For the kinetic resolution of racemic a-acetoxyamide 6 several native enzymes were used (Scheme 5.5). The native Upases from Pseudomonas cepacia (PCL) and porcine pancreas (PPL) showed the highesL although stiU unsatisfactory, enantios-electivity ( = 5.1 and 3.5, respectively). Upon immobilization into a solgel matrix, the enantioselectivity of PCL was improved significantly to 30.5. The covalent immobilization on Eupergit increased the enantioselectivity even more (34.0) [23]. 

The NHase responsible for aldoxime metabolism from the i -pyridine-3-aldoxime-degrading bacterium, Rhodococcus sp. strain YH3-3, was purified and characterized. Addition of cobalt ion was necessary for the formation of enzyme. The native enzyme had a Mr of 130000 and consisted of two subunits (a-subunit, 27 100 (3-subunit, 34500). The enzyme contained approximately 2 mol cobalt per mol enzyme. The enzyme had a wide substrate specificity it acted on aliphatic saturated and unsaturated as well as aromatic nitriles. The N-terminus of the (3-subunit showed good sequence similarities with those of other NHases. Thus, this NHase is part of the metabolic pathway for aldoximes in microorganisms. 

In the case of the phosphotriesters 94, the use of engineered mutants of phosphotriesterase allowed not only to enhance but even to reverse the stereoselectivity of the native enzyme. The ees of the recovered esters exceeded 95%. 

Mutagenesis of known enzyme towards a desired activity will be the fastest developing direction. The use of mutants of simple serine-hydrolases, which exhibit the phosphotriesterase activity (in contrast to the native enzymes, which are irreversibly inhibited under such conditions), clearly shows that practically any kind of substrates can be enzymatically transformed. The... 

If the proton-donating ability of the amino acid at 188 is weaker, then the enantioselectivity of the reaction will be reversed compared to that of native enzyme. As shown in Table 3, the absolute configuration of the products by this mutant is opposite to those of the products obtained by the native enzyme and the ee of the products dramatically increased to 94 and 96%, respectively. This inversion of the enantioselectivity of the reaction supports the reaction mechanism that the Cys 188 of the native enzyme is working as the proton donor to the intermediate enolate form of the product. ... 

Further indications for an additional subunit were provided by a crosslinking analysis of C Eg solubilized H,K-ATPase, which exhibited ATPase and phosphatase activities, and ligand affinities comparable to the native enzyme [70]. Glutar-aldehyde treatment of soluble protein fractions resolved on a linear glycerol gradient revealed no active fraction enriched in monomeric (A/p = 94 kDa) H,K-ATPase. Instead, K -ATPase activity was only obtained in fractions enriched in particles of Mr = 175 kDa. This size also suggested that the functional H,K-ATPase unit is a heterodimer of a catalytic subunit and an additional subunit, since the apparent molecular mass of 175 kDa is probably too small to be a homodimer of the catalytic subunit. 

The approximate location of the epitopes for more than 40 monoclonal anti-ATPase antibodies has been mapped to various regions within the cytoplasmic domain of the Ca " -ATPase [285,302-304]. All antibodies were found to bind with high affinity to denatured Ca -ATPase, but the binding to the native enzyme showed significant differences depending on the location of antigenic sites within the ATPase molecule. 

Of the 20 residues that react with A-ethylmaleimide in the non-reduced denatured Ca -ATPase at least 15 are available for reaction with various SH reagents in the native enzyme [75,239,310]. These residues are all exposed on the cytoplasmic surface. After reaction of these SH groups with Hg-phenyl azoferritin, tightly packed ferritin particles can be seen by electron microscopy only on the outer surface of the sarcoplasmic reticulum vesicles [143,311-314]. Even after the vesicles were ruptured by sonication, aging, or exposure to distilled water, alkaline solutions or oleate, the asymmetric localization of the ferritin particles on the outer surface was preserved [311,313,314]. 

Neumann A, A Siebert, T Trescher, S Reinhardt, G Wohlfarth, G Dickert (2002) Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans substrate specificity of the native enzyme and its cor-ronoid cofactor. Arch Microbiol 111 420-426. 

Metabolic and enzyme engineering have received a lot of attention in academic institutions and are now being applied for the optimization of biocatalysts used in the production of a diverse range of products. Engineered microorganisms, even with non-native enzyme activities, are being used for novel products and process improvements for the production of precursors, intermediates and complete compounds, required in the pharmaceutical industry (Chartrain et ai, 2000). 

These data demonstrate that both GSH and GSSG have profound effects on Na/K ATPase activity and may act in concert to modify enzyme activity during oxidant stress. However, it should be recognized that the steric conformation of an isolated enzyme preparation in a chemically buffered solution may be considerably different to the native enzyme located in a dynamic lipid bilayer. For this reason, these investigations have been extended to include a variety of preparations in which the Na/K pump is in its native environment. 

Villamiel, M. and Jong, P. D. (2000b). Influence of high-intensity ultrasound and heat treatment in continuous flow on fat, proteins, and native enzymes of milk. /. Agric. Food Chem. 48, 472-478. 

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... 

Coordinates of the SGPA active site residue and of the two water molecules W184 and W210 are from a further refinement of the published (1.8 A resolution) structure (Ref. 118b) with a resolution extending beyond 1.7 A for the native enzyme James, M. N. G, Sielecki, A. R. Private communications (1983, 1984)... 

Finally, in the case of inhibitory substrate analogues such as allo-xanthine, strong evidence has recently been presented that these bind to molybdenum in reduced xanthine oxidase (33). If the enzyme is reduced with xanthine, then treated anaerobically with alloxanthine and finally exposed to air, catalytic activity is lost. Though flavin and iron in the final product are in the oxidized state, there are significant spectral differences between it and the native enzyme. These are believed (33) due to reduction of molybdenum from Mo(VI) to Mo(IV) and complexing of... 

Fig. 6. Difference spectra between xanthine oxidase inactivated with various pyra-zolo [3, 4-d] pyrimidines and the native enzyme. The spectra are believed to represent the increase in absorption occurring when Mo(VI) of native enzyme is converted to Mo(IV) complexed with the inhibitors. Spectra were obtained by treating the enzyme with inhibitors in the presence of xanthine, then admitting air, so as to re-oxidize the iron and flavin chromophores. The extinction coefficients, de, are expressed per mole of enzyme flavin. Since some inactivated enzyme was present, extinction coefficients per atom of molybdenum of active enzyme will be about 30% higher than these values. (Reproduced from Ref. 33, with the permission of Dr. V. Massey.)...

Fungal cutinases show no free SH groups but have 4 Cys residues, indicating that they are in disulfide linkage [119]. The reaction of the native enzyme with DTE was extremely slow but in the presence of SDS at its CMC rapid reduction could be observed [102]. Reduction of the disulfide bridge resulted in irreversible inactivation of the enzyme and the protein tended to become insoluble. CD spectra of cutinase in the 205-230 nm region, before and after DTE reduc-... 

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