Organic cofactors

Dioxygenases often have broad substrate specificity and require only a minimal characteristic structure for substrate recognition [310], Transition metal or an organic cofactor mediates dioxygen activation needed by the oxygenases action. Iron and copper, in their lower oxidation states are the metals most commonly used, but also organic co-factors like dihydroflavin and tetrahydropterin are able to activate the oxygen molecule. 

Enzymes containing amino acid radicals are generally associated with transition metal ions—typically of iron, manganese, cobalt, or copper. In some instances, the metal is absent it is apparently replaced by redox-active organic cofactors such as S -adenosylmethionine or flavins. Functionally, their role is analogous to that of the metal ion in metalloproteins. 

ENGINEERING METAL INSERTION INTO ORGANIC COFACTORS... 

As we will see in subsequent chapters, many metalloproteins have their metal centres located in organic cofactors (Lippard and Berg, 1994), such as the tetrapyrrole porphyrins and corrins, or in metal clusters, such as the Fe-S clusters in Fe-S proteins or the FeMo-cofactor of nitrogenase. Here we discuss briefly how metals are incorporated into porphyrins and corrins to form haem and other metallated tetrapyrroles, how Fe-S clusters are synthesized and how copper is inserted into superoxide dismutase. 

Engineering Metal Insertion into Organic Cofactors.30... 

Synthesis of metal-organic cofactors Molybdopterin-cofactor in dehydrogenases FeMo-cofactor for nitrogenase... 

Covalent insertion of metal-organic cofactors Cytochrome c heme-ligase... 

Coenzymes are densely functionalized organic cofactors capable of catalyzing numerous diverse chemical reactions. Nature exploits the intrinsic chemical reactivity of these molecules to extend the chemical fimctionaUty of enzymes well beyond the reactivity of the coded amino acids. When these constituents are incorporated via covalent or non-covalent interactions into coenzyme-depen-dent enzymes, the inherent reactivity of the co enzyme is augmented and directed to effect chemical transformations with substrate and product selectivities, rates, and yields that are unachievable by either the protein or coenzyme alone. Thus, coenzymes play a critical role in the execution of a large number of essential metabolic processes. 

Tetranuclear iron-sulfur clusters are key relay stations in the electron flow in photosynthesis. Photosystem I comprises three subunits, PsaA, PsaB and PsaC. The latter contains two [Fe4S4] centres FA and FB. The core subunits PsaA and B, respectively, house a [Fe4S4] centre denoted FX in addition to other, organic cofactors. The role of this latter cluster was probed in preparations partially devoid of PsaC. It was concluded from the results that FX has a major role in controlling the electron transport through PS I.236 Since the final acceptor of the electrons in PS I is a ferredoxin with a [Fe2S2] cluster it was of interest to study a... 

Figure 2 Molecular structures and IUPAC numbering scheme of organic cofactors occurring in photosynthetic reaction centres (bRC, PS I, PS II). (Bac-teriolpheophytin is the free base of (bacterio)chlorophyll plastoquinone (PQ) is found in PS If phylloquinone or vitamin K, ( VK,) in PS I many bacteria contain ubiquinone (UQ). Shown is also the amino acid tyrosine (Tyr, Y) that is redox active in PS II.

Topaquinone (TPQ). Both bacteria and eukaryotes contain amine oxidases that utilize bound copper ions and 02 as electron acceptors and form an aldehyde, NH3, and H202. The presence of an organic cofactor was suggested by the absorption spectra which was variously attributed to pyridoxal phosphate or PQQ. However, isolation from the active site of bovine serum... 

The recognition that the Mo in the molybdoproteins exists in organic cofactor forms came from studies of mutants of Aspergillus and Neurospora.650 In 1964, Pateman and associates discovered mutants that lacked both nitrate reductase and xanthine dehydrogenase. Later, it was shown that acid-treated molybdoenzymes released a material that would restore activity to the inactived nitrate reductase from the mutant organisms. This new coenzyme, a phosphate ester of molybdopterin (Fig. 15-17), was characterized by Rajagopalan and coworkers.650 651 A more complex form of the coenzyme, molybdopterin cytosine dinucleotide... 

It is not known at present if the nickel is coordinated directly to the protein, as in copper and iron-sulfur proteins, or to an organic cofactor, as in the molybdenum hydroxylases and hemoproteins. 

Copper-dependent amine oxidases contain a type II copper (83-85). The yellow-pink color of amine oxidase implies the existence of another organic cofactor whose identification has not yet been unambiguously... 

Eschenmoser [15] has recently alluded to the concepts of structure-directed synthesis and autochthonous reactivity — the propensity of certain systems to undergo reactions as a consequence of their intrinsic molecular structural factors — within the context of certain biomolecules, particularly the organic cofactor, vitamin B12. The formation of the AD ring junction in the corrin ligand had to be regarded as a major hurdle in any proposed chemical synthesis of cobyric acid (5) (Fig. 2), and hence of vitamin B12. However, driven initially by the desire to identify a dark, and therefore potentially biomimetic, variant of the photochemical... 

Cofactors. Many enzymes require relatively loosely bound organic cofactors (coenzymes) or metal ions for activity. A biochemist must be alert for indications of such a requirement during purification of an enzyme, because it may be necessary to add such cofactors to the assay solution to obtain enzyme activity. The interactions of coenzyme with enzyme can be analyzed by kinetic and spectral techniques. Similar techniques are used to evaluate interactions with metal ions. 

Many enzymes require cofactors. When such cofactors are metal ions (e.g. Cu2+, Zn2+, Ni2+, Fe2+/Fe3+) the enzymes are called metalloenzymes. When organic cofactors (coenzymes) are required the coenzyme may be free or tightly bound to the enzyme (as a so-called prosthetic group ). The enzyme-cofactor complex is termed the holoenzyme and the enzyme free of cofactor or coenzyme is called the apoenzyme . 

The nature of oxidative processes requires the removal of dectrons from the substrate and many enzymes of the redox class contain transition metals which act as an electron sink." Those enzymes which do not satisfy this requirement need organic cofactors such as nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide 2 -phosphate to act as dectron accqitors, although simple qui-nones have been shown to suffice. ... 

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