Enzyme reconstitution

Electroosmotic flow, 195 End column detection, 89 Energy barrier, 16 Enzyme electrodes, 172, 174 Enzyme immunoassays, 185 Enzyme inhibition, 181 Enzyme reconstitution, 178 Enzyme wiring, 178 Equilibrium potential, 15 Ethanol electrodes, 87, 178 Exchange current, 14... 

The presence of calcium in horseradish peroxidase was demonstrated originally by Haschke and Friedhoff, working with the C and A (imspec-ified, but likely to have been predominantly A2) isoenzymes (209). HRP C and HRP A contain 2.0 0.13 and 1.4 0.19 moles calcium per mole enzyme, respectively, as determined by atomic absorption spectroscopy. Incubation in 6 M guanidinium hydrochloride and 10 mM EDTA for 4 hours at neutral pH and room temperature gave calcium-depleted enzymes with specific activities decreased by 40% and 15%, respectively. The thermal stability of calcium-depleted HRP C was also reduced compared to native enzyme. Reconstitution was successful only with calcium-depleted HRP C (209). It remains to be established whether this reflects true structural differences between the calcium binding sites of the two isoenz5unes, or is a consequence of the relatively harsh... 

During this same period Suzuki et al. (28-31) published a series of papers on the properties of yeast aconitase purified from Candida lipotytica. This material remained active upon purification and was analyzed to contain 2 Fe and 1 S=/protein molecule of 68,500 daltons (28). One Fe could be removed with chelators without loss of activity (30). Enzyme reconstituted with 57pe was studied with EPR and MSssbauer spectroscopy (31). Even though the measured Mbssbauer parameters did not match those of other Fe-S proteins ( AEq = 0.9 mm/s and 5 = 0.36 mm/s for the dominant species), the spectra were interpreted as resulting from a [2Fe-2S] cluster. In addition chemical analyses on the reconstituted material now gave 2 Fe... 

Coenzyme and substrate analogs. The structures of enzyme NAD1 substrate complexes (Fig. 15-3) may be studied by X-ray crystallography under certain conditions or can be inferred from those of various stable enzyme-inhibitor complexes or from enzyme reconstituted with NAD+ that has been covalently... 

If E. coli is grown in the presence of relatively high concentrations (3.5 fjt,M) of 60Co2+, an active 60Co(II) enzyme is synthetized (122). Its properties resemble those of the Co(II) enzyme reconstituted from the apoenzyme. Certain mutants of E. coli produce defective alkaline phosphatases. The Co(II) enzyme from the mutant U47 lacks the characteristic bands of the Co (II)-complex of the normal enzyme, and the spectrum is broad and poorly resolved (123). 

Fig. 2.20 Formal fitness landscape of various biocatalytic and inactive (i.e., the latter being located outside the window of essentiality ) metal ions for the carboxypeptidase A (Vallee and Williams 1968). The enzyme reconstituted by cobalt (treatment with EDTA, then addition of Co +) is considerably more active than the native Zn version , while Cd (and other ions) afford poorly to non-active metalloproteins in this case. (Relative) catalytic turnover rates are from Vallee and Williams 1968, x and c values this work and previous publications by this author. Here, sufficient abundance of the corresponding ions is taken for granted owing to in-vitro addition to apoprotein, so c k is not hmited by c, but merely represents the catalytic features pertinent to k, obtained from either biochemistry (several metal ions being present, e.g. in phosphatases) or experiments with reconstituted apoproteins...

Enzymes reconstituted with semi-artificial cofactors linked to redox relays... 

Photochemical Activation of Semi-synthetic Enzymes Reconstituted with Non-natural Photosensitive Cofactors... 

K.M., and Spiro, T.G. (1993) Complete assignment of cytochrome c resonance Raman spectra via enzymic reconstitution with isotopically labeled hemes. Journal of the American Chemical Society, 115, 12446-12458. 

NEM <2> (<2> inhibits phosphoenol-dependent phosphorylation of mannitol and mannitol 1-phosphate, no effect on activity of exchange reaction [7] <2> enzyme reconstituted in phospholipid vesicles [11]) [7, 11]... 

The protein was expressed in E. coli using an E. co//-optimized cDNA [2670]. The pmified enzyme, reconstituted with recombinant adreno-doxin and NADPH-adrenodoxin reductase, did not catalyze the oxidation of cholesterol, vitamin Dj, la-hydroxy vitamin D3, or 25-hydroxyvita-min D3. In other studies, none of a test set of pro-carcinogens [350] was activated to a genotoxic product. 

In spite of the difference in prosthetic groups, the catalytic activity of TPN-cytochrome c reductase is essentially identical with that of the yeast enzyme. In the presence of excess TPN, reduced with hexosemonophosphate and glucose-6-phosphate dehydrogenase (Zwischenferment) the turnover number was found to be 1140 moles of cytochrome per mole of flavoprotein per minute with the liver enzyme and 1300 with the yeast enzyme. Cytochrome c reductase from liver also resembles the enzyme from yeast in the behavior of the protein component with FAD and FMN (Table 3). In each case the enzyme reconstituted with FMN is more active than with FAD, despite the fact that the natural prosthetic group of the liver enzyme is FAD. 

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