Holo-enzymes

A in our group [26,29]. Both structures were of the holo-enzyme, i.e., the enzyme in presence of the cofactor. 

For those investigated, PQQ-containing dehydrogenases require a divalent metal ion for activity, Mg in one case and Ca in all other cases (see Table 1). For some enzymes, Ca removal leads also to detachment of PQQ, providing an apo enzyme that can be easily reconstituted by addition of PQQ and Ca. For other enzymes, removal of the metal ion plus PQQ is not so easy, but fortunately several bacteria produce apoenzyme gratuitously as they are still able to produce the protein part of the dehydrogenase, but not PQQ (at least not under the laboratory conditions used for cultivation). Also in these cases, reconstitution to holo enzyme is easy, in vitro as well as in vivo the latter leads to active enzyme coupled to the respiratory chain. However, indications exist that a special protein is required for inserting Ca in the case of MDH [58],... 

The UV-VIS spectrum of 100 p,M VCPO at pH 8.3 is displayed in Figure 1.7a, which shows the spectra of apo-, holo-, and holo-enzyme after the addition of the first substrate H202 (peroxo-intermediate) (Renirie et al. 2000a). If a halide is added to the peroxo-intermediate, the holo-spectrum is reformed, in line with the scheme shown in Figure 1.6. 

Figure 1.7 (a) UV-VIS absorption spectra of 100 /vanadium chloroperoxidase at pH 8.3. The lower solid line shows the apo-enzyme, the upper solid line the holo-enzyme after addition of 100 /xM vanadate. The dotted line is the peroxo-form of the enzyme after addition of 100 /optical absorbance at 316 nm ( ). Absorbance of free vanadate under the same buffer conditions (O). Reprinted with permission from Renirie, R., Hemrika, W., Piersma, S.R. and Wever, R. (2000). Biochemistry, 39, 11 33-1141. Copyright 2000 American Chemical Society. 

Interaction with Adenosylcobalamin. It has been considered generally that adenosylcobalamin or its analogs binds to the apoprotein of diol dehydrase or other adenosylcobalamin-dependent enzymes almost irreversibly (4). However, we found that the holo-enzyme of diol dehydrase was resolved completely into intact apoen-zyme and adenosylcobalamin when subjected to gel filtration on a Sephadex G-25 column in the absence of K+ (9, 10). Among the inactive complexes of diol dehydrase with irreversible cobalamin inhibitors, those with cyanocobalamin and methylcobalamin also were resolved upon gel filtration on Sephadex G-25 in the absence of both K+ and substrate, yielding the apoenzyme, which was reconstitutable into the active holoenzyme (II). The enzyme-hydroxocobalamin complex, however, was not resolvable under the same conditions. The enzyme-cobalamin complexes were not resolved at all by gel filtration in the presence of both K+ and substrate. When gel filtration of the holoenzyme was carried out in the presence of K+ only, the holoen-... 

Figure 4 UV-VIS absorption spectra of lOOpM chloroperoxidase at pH 8.3. Holo-enzyme was obtained by addition of 100 pM vanadate to the apoenzyme and the peroxo form by addition of 100 pM H2O2 to the holo-enzyme. (Reprinted with permission from Ref. 35. 2000 American Chemical Society)...

Activators can only elicit a very low level of activation in a transcription system reconstituted from pure RNA polymerase 11 (pol 11) and basal factors, some types of co-factor are needed in addition. One such factor was found and termed Mediator. In an in vitro transcription system reconstituted from essentially pure factors, the mediator complex was shown to stimulate basal transcription 50-fold and to support transcriptional activation from the Gal4-VP16 and Gcn4 activating protein. The Mediator complex could also be purified in a complex with RNA polymerase II (pol II), thus forming a holo-enzyme form of pol II. 

A high degree of structural specificity for inhibitor-enzyme interaction was demonstrated in studies where several isoquinoline and pyridine derivatives were tested with the reductase (150—155). The partially competitive relationship between inhibitor and dithiol substrate and the specificity of inhibitor binding are consistent with a model in which an inhibitor-iron complex binds, or free inhibitor coordinates with holo-enzyme, at or adjacent to a site normally occupied by dithiol substrate. Studies of this class of inhibitors have not only served as a probe into the structure of the mammalian reductases but also as a measure of differ-... 

The availability of sufficient quantities of enzymes for crystallization studies has led to the crystal structures been obtained for several dehydrogenases. For example, two tetrameric NADP+-dependent bacterial secondary alcohol dehydrogenases from the mesophilic bacterium Clostridium beijerinckii and the thermophilic bacterium Thermoanaerobium brockii have been crystallized in the apo- and the holo-enzyme forms, and their structures are available in the Protein Data Bank11451. The crystal structure of the alcohol dehydrogenase from horse liver is also available[40 21. 

A key property of the enzyme, established by x-ray data, is the existence of two protein domains in each monomer that are relatively free to rotate relative to each other. The apo- and holo-enzymes exist in the so-called open form, whereas binding of NADH coenzyme induces rotation of one domain, resulting in the so-called closed form - " (Figures 2.34 and 2.35). Closure brings the catalytic zinc ion into an ideal position to bind the aldehyde substrate in such a way that the reactive CH2 group of the nicotinamide ring of NADH points toward... 

An understanding of the mode of binding of the co-enzyme to the apo-enzyme is also required. The ability of various compounds, structurally related to pyridoxal phosphate, either to combine with the apo-enzyme to form an active holo-enzyme or to inhibit the formation of an active holo-enzyme, can be used to assess the affinities of these substances for the apo-enzyme. Such studies indicate that the phosphorylated 5-hydroxymethyl group of pyridoxal plays a major role in the binding of pyridoxal phosphate to the apo-enzyme, and that the free phenolic group also contributes to the binding ". 

Inhibition by Inactivation or Displacement of the Co-enzyme The function of pyridoxal phosphate as the co-enzyme of histidine decarboxylase depends on the ability of the aldehyde group to react with the a-amino group of histidine. However, the aldehyde group also reacts with a wide variety of amino acids, amines and carbonyl reagents. The specificity of the holo-enzyme-substrate interaction is therefore due to the apo-enzyme. The co-enzyme attached to the active centre is presumably in equilibrium with free co-enzyme in the surrounding solution. 

The sulfide ligands of the cluster are also expected to attract protons. The Bronsted basicity of the extracted FeMoco has been evaluated using the kinetic method described in Section 8.22.2.3.4, suggesting that the first of the cluster is about 8. A similar p/fa of 9 comes from the pH variation in rates of H2 evolution and acetylene reduction by the holo enzyme. The protonation of the FeMoco also has been evaluated with ab initio calculations, showing that, as expected, the //2-sulfides should be more basic than the //3-sulfides. This work showed the best proton binding position to be the center of the FeMoco, undoubtedly a result of the omission of the central X atom in the theoretical model. The sulfur atoms of synthetic FeS clusters can also act as bases, and the protonated forms may play a role in redox chemistry. ... 

FIG. 1. Interrelationships between vitamin B6 and phosphorylase metabolism. The low rate of turnover of glycogen phosphorylase (gpb) and the lack of exchange of free and protein-bound PLP mean that exchange into the muscle pool is largely controlled by the kinetics of turnover of the enzyme. At present, it is not known whether resolution of the holo-enzyme is a prerequisite or consequence of phosphorylase degradation. Reproduced with permission of llie Biochemical Journal. 

From the synthetic chemistry perspective it is not an easy task to stabilize a monomeric Mo (W is even more difficult) complex in the +v or +iv oxidation state but in biological reactions the participation of Mo (or W) in these oxidation states is routine and takes place easily. In Nature the readily available form of Mo or W is the M04 ion. Details of the incorporation of these metals into apo-enzymes to develop holo-enzymes is not in the focus of this review but certain qualification of these elements related to their chemistry should be highlighted. It is rather difficult to reduce M04 " in basic to... 

---