Apoenzyme model

Abstract. The inclusion behavior of the octopus cyclophane constructed with a rigid macrocyclic skeleton and eight hydrocarbon chains was studied in aqueous media by means of fluorescence and electronic absorption spectroscopy. Both hydrophobic and electrostatic interactions came into effect in the host-guest complexation process. The cyclophane acted as an effective apoenzyme model for constitution of an artificial vitamin B -dependent holoenzyme by simultaneous incorporation of pyridoxal-5 -phos-phate and a hydrophobic alkylammonium substrate into the host cavity to give the Schiff-base species, showing the substrate selectivity. 

In conclusion, it became clear that the octopus cyclophane can be utilized as an effective apoenzyme model for constitution of an artificial vitamin B -dependent holoenzyme. The ternary complex is formed with 1, PLP, and a substrate in the initial reaction stage, and then the latter two species bound to 1 undergo Schiff-base formation. Molecular recognition is exercised by the octopus cyclophane in favor of hydrophobic substrates. 

The finding that thiamine, and even simple thiazolium ring derivatives, can perform many reactions in the absence of the host apoenzyme has allowed detailed analyses of its chemistry [33, 34]. In 1958 Breslow first proposed a mechanism for thiamine catalysis to this day, this mechanism remains as the generally accepted model [35]. NMR deuterium exchange experiments were enlisted to show that the thiazolium C2-proton of thiamine was exchangeable, suggesting that a carbanion zwitterion could be formed at that center. This nucleophilic carbanion was proposed to interact with sites in the substrates. The thiazolium thus acts as an electron sink to stabilize a carbonyl carbanion generated by deprotonation of an aldehydic carbon or decarboxylation of an a-keto acid. The nucleophilic carbonyl equivalent could then react with other electro-... 

The ionization state of the coenzyme is also important. During reduction a charged pyridinium species is created while during oxidation the charge is lost. Thus, more polar environments favor reduction while more hydrophobic conditions favor oxidation [69]. Therefore the apoenzyme environment and model system scaffolds must not only enhance the reactivity of the coenzyme, but must also address these issues of equilibrium and stability. 

Fig. 4. Structure of molybdopterin and MoCo and its suggested attachment to plant NR apoenzyme. This model is adapted from Kramer et al. (1987) for the molybdopterin, Gardlik Rajagopalan (1990) for the pterin reduction state (shown here as a 5,6-dihydropterin, one of the three possible structures) and Neame Barber (1989) for the hypothetical thiol ligand from the side chain of Cysl80 of tobacco NR. A, Molybdopterin. B, MoCo.

An early model for the role of the apoenzyme in coenzyme Bi2-dependent mutase... 

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. 

Support for a concerted model for the yeast enzyme has come from X-ray small angle scattering experiments (162) as well as from hydro-dynamic and optical rotation studies (163, 164). A. volume contraction of about 5% occurs on binding of NAD to the apoenzyme, presumably related to tightening of the tetramer and expulsion of water mojecules. The relation between NAD bound (R) and change of volume (Y) was hyperbolic, in accord with the concerted model. It was lator shown (166) from buoyant density and preferential hydration studies that water is indeed excluded from the yeast enzyme on binding to NAD, such that a volume contraction of about 6% occurs. Furthermore, fluorimetric and calorimetric titrations over the range 6°-40° showed independence of... 

Because of these clear differences of chemical pathway in the enzymatic and model reactions, it is difficult to assess the actual factor by which the apoenzyme enhances the reactivity of reduced flavin with O2. It is fairly clear, however, that the environment of the apoenzyme suppresses homolytic pathways and, presumably by general-acid-base and steric mechanisms, allows only heterolytic bond cleavages. 

The Z isomer of the model substrate 4-tra s- N,]V -dimethylamino)cinnamaldoxime (Z-DMOX) forms a ternary complex with NAD and LADH. The Co", Ni, Cu and Cd enzymes (with the metal substituted for the catalytic zinc) and the apoenzyme also form ternary complexes with Z-DMOX. The affinity of the apoenzyme-NAD complex for Z-DMOX is much lower and the rate of Z-DMOX dissociation from the apoenzyme complex was 10 -fold greater than the rates found for the metal-substituted enzymes. Complex formation results in a red shift (43 to 83.5 nm) in the DMOX UV-visible spectrum, due, it is suggested, to bonding of the oxime nitrogen to a strong electrophilic centre, either the Zn or the nicotinamide ring of NAD, a view that is compatible with the known structural features of the LADH/NADH/MejSO complex. The high affinities and slow rates of dissociation of the metal-substituted enzyme complexes are attributed to the coordination of Z-DMOX to the active site metal. ... 

Jones manually derived positions of amino acid residues in the apoenzyme structure and some ternary complexes published before 1977. Figure 21 shows a comparison between the present results with the earlier definition. The boundary region of model (co13) has also been assigned in the Figure. Ser-48, Phe-93 and lle-318 are in the same location while Leu-57 is in layers 3, 4 and 5 in our X-ray derived model. Using the present method positions of residues were positioned more accurately. 

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