Apoenzyme, preparation

Apoenzyme prepared from muscle holoenzyme by treatment with charcoal is unstable and diflScult to crystallize (60, 61). Consequently, it has not so far been possible to solve the three-dimensional structure of apo-GAPDH by X-ray crystallographic methods. Suzuki and Harris (18) were able to prepare stable crystals suitable for X-ray diffraction analysis of both holo- and apoenzyme from the thermophile B. stearother-mophilus. GAPDH from this source is considerably more stable than enzyme from mesophiles (17, 18), and this stability is retained even in the absence of NAD (Fig. 9). Wonacott and colleagues (62, cf. 18) have shown that these holoenzyme crystals are orthorhombic with space group P2,2i2 the unit cell, like that of the lobster muscle enzyme, consists of four tetramers. Apoenzyme crystals were found to be monoclinic (space group P2i), and the unit cell consists of two tetramers. 

The effect of edta on alkaline phosphatase from E. coli and on the cobalt(ii) and copper(ii) derivatives of the protein has been studied by the measurement of enzyme activity and bye.s.r. spectroscopy. From dialysis experiments on an edta-contaminated apoenzyme, it was found that the edta binds to the metal-free protein. Furthermore, in the complete absence of edta only two Zn + or Co + ions per enzyme molecule were required for full enzyme activity and it is suggested that reports in the literature that more than two metal (ons are necessary (four is the number commonly quoted) may be explained by varying levels of edta contamination in the enzyme and apoenzyme preparations. The ligand edta also affects the e.s.r. spectrum of copper alkaline phosphatase, thus accounting for the two types of signal reported previously and their different behaviour towards phosphate. 

Titration with chelators of a metalloenzyme preparation from which extraneous metals and chelators have been removed produces a characteristic enhancement of the intrinsic protein fluorescence (excitation at 280 nm, emission at 350 nm) (13). This fluorescence enhancement by nonfluorescent chelators is instantaneous, reversible by excess added divalent metal ions, and can occur without loss of activity. Different chelators give different characteristic amounts of fluorescence enhancement at saturation, demonstrating the specific effect of the chelator on the fluorescence of the apparent metalloenzyme-chelator complex. In contrast, if the effect of chelators were simply to complex with Mg2 after its dissociation from the metalloenzyme, the resulting apoenzyme should have identical fluorescence properties regardless of which chelator was utilized. 

The flavoenzyme D-lactate dehydrogenase from yeast has been reported to contain zinc (134). An apoenzyme can be prepared and reactivated by Zn2+ or Co2+ (135). When yeast is grown in the presence of added Co2+, a Co(II) enzyme is synthesized. The biosynthetic Co(II) enzyme was found to have different catalytic properties compared to the enzyme reactivated from the apoenzyme (136). Only rather fragmentary data have been published on this subject, and the differences in cobalt binding obtained by the two methods of preparation are unknown. 

A model of a flavin-based redox enzyme was prepared.[15] Redox enzymes are often flavoproteins containing flavin cofactors flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). They mediate one- or two-electron redox processes at potentials which vary in a range of more than 500 mV. The redox properties of the flavin part must be therefore tuned by the apoenzyme to ensure the specific function of the enzyme. Influence by hydrogen bonding, aromatic stacking, dipole interactions and steric effects have been so far observed in biological systems, but coordination to metal site has never been found before. Nevertheless, the importance of such interactions for functions and structure of other biological molecules make this a conceivable scenario. 

Reaction of the apoenzyme with various metal ions allows the preparation of other metallocarbonic anhydrases and the Cu(II), Co(II) and Co(III) enzymes have been prepared. The cobalt(II) enzyme shows some catalytic activity. The d-d spectrum of the cobalt(II) derivative, which is high spin, is markedly pH dependent (Fig. 5-19). A plot of the molar absorbance at 640 nm versus pH does not follow a simple pattern, as expected for a single ionising group, but is consistent with two ionising groups with pK values of < 6 and > 7. 

Ferricyanide appears to accept electrons from both the flavin and the heme (299-302), and it is believed that heme is required for cytochrome c reduction. Forestier and Baudras (302) have reported that, by treatment with guanidinium chloride, preparations of cytochrome 62 could be rendered partially deficient in flavin and heme. Thus, enzyme preparations were obtained which contained 65-75% flavin and variable amounts of heme from about 12 to 100%. The low heme preparations showed considerably greater loss of cytochrome c reductase than ferricyanide reductase activity. When preparations with increasing content of heme relative to flavin were tested, both the ferricyanide and the cytochrome c reductase activities increased as a linear function of heme to flavin ratio (up to heme flavin =1), but the increase in the heme content had a much greater effect on the cytochrome c reductase activity of the enzyme. The apoenzyme of cytochrome 62 has been prepared. However, reconstitution with FMN, heme, and FMN plus heme in all cases resulted in extremely... 

The molecular weight of cytochrome c peroxidase has been determined to be 34,100 on the basis of a sedimentation constant of 3.55 S, a diffusion constant of 9.44 F, and a partial specific volume of 0.733 ml/g (4 )-The enzyme exists as a monodisperse monomer containing one ferric protoporphyrin IX, which is noncovalently bound (/, , 14). No other transition metal is detected in crystalline preparations of the enzyme (22). The apoenzyme moiety is an acidic protein with an isoelectric point at pH... 

Use of apoenzymes for the detection of metal ions Generally, apoenzymes of metalloenzymes can be used for the detection of the corresponding metal ion. Restoration of enzyme activity obtained in the presence of the metal ion can be correlated to its concentration. This principle has been demonstrated in the detection of copper while evaluating reconstituted catalytic activities in galactose oxidase and ascorbate oxidase and also in the detection of zinc since this ion is essential for the activity of carbonic anhydrase and alkaline phosphatase [416]. The need of stripping the metal for the preparation of the apoenz5une may demand tedious procedures and a catalytic assay with the addition of the substrate is always required for detection. 

Much attention has been paid to metal-substituted alkaline phosphatases, notably Co" d-d spectra), Mn" (ESR) and Cd ( Cd NMR). The apoenzyme may be prepared by use of ammonium sulfate to remove zinc. After about five days the apoenzyme may be isolated having less than 3% of the original zinc. Furthermore, the apoenzyme is uncontaminated by chelating agents, which show a tendency to bind to the apoenzyme. A range of metalloalkaline phosphatases may be prepared from the apoenzyme. The binding of cadmium at three separate sites can be confirmed by the use of " Cd NMR, which shows " three separate resonances at 153, 72 and 3 p.p.m. in the phosphorylated dimer Cd"6AP. When all three sites are occupied by Cd , the enzyme has a very low turnover, at least 10 times slower than the native Zn" enzyme. This slow turnover number has made the Cd" enzyme particularly useful in NMR studies. 

This enzyme is monomeric, consisting of one polypeptide apoenzyme and one prosthetic group, a flavin adenine dinucleotide (FAD) moiety (Fig. 2.1), that is nonco-valently bound, with an association constant1 of 3.6 x 106 M. The active holoenzyme can be prepared, or reconstituted, by the addition of FAD to a solution containing the apoenzyme. 

Shoolingin-Jordan PM, Warren MJ, Awan SJ. Dipyrroraethane cofactor assembly of porphobilinogen deaminase formation of apoenzyme and preparation of holoenzyme. Methods Enzymol 1997 281 327-36. 

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