Apoenzyme, protein-chemical

Protein-chemical Properties of Apoenzyme. Adenosylcobalamin-dependent diol dehydrase was discovered and isolated first by Abeles and co-workers (3, 4) in the cells of Klebsiella pneumoniae (formerly known as Aerohacter aero genes) ATCC 8724 grown without aeration in a glycerol or glycerol-1,2-propanediol medium. This enzyme catalyzes the conversion of 1,2-propanediol, 1,2-ethanediol, and glycerol to propionaldehyde, acetaldehyde, and j3-hydroxypropionaldehyde, respectively (4, 5). Adenosylcobalamin and K+ or other monovalent cations of a similar size are required for catalysis. Recently, the au-... 

Cofactor is a non-protein chemical compound that facilitates the activity of an enzyme and is required for the biochemical reaction to occur. A further biochemical classification exists for the enzyme and its cofactor. The enzyme without its cofactor is an apoenzyme the enzyme together with its cofactor is called an oloenzyme. 

Enzymes are large protein molecules (apoenzymes), which act as catalysts for almost all the chemical reactions that occur in living organisms. The structures of a number of enzymes contains groups of metal ions, known as metal clusters, coordinated to the peptide chain. These enzymes are often referred to as metalloenzymes. Many enzymes require the presence of organic compounds (co-enzymes) and/or metal ions and inorganic compounds (co-factors) in order to function. These composite active enzyme systems are known as holoenzymes. 

Carboxypeptidase A is one of the most intensely investigated zinc metalloenzymes. The enzyme as isolated contains 1 g-atom of zinc per protein molecular weight of 34,600. Removal of the metal atom either by dialysis at low pH or by treatment with chelating agents gives a totally inactive apoenzyme (46). Activity can be restored by readdition of zinc or one of a number of other di-valent metal ions (47). Through a combined use of chemical modification and transient state kinetic studies, it has been possible to determine the role of zinc in the catalysis of ester and peptide hydrolysis by this enzyme. 

The metal constitutes a reactive group of the enzyme. It represents a tag which has the chemical features typical of the metal in question and similarly entails to the enzyme attributes which allow of precise measurement of the apoenzyme, coenzyme, and substrate interaction by techniques characteristic of the study of metal complexes in simpler systems (Calvin, 1954). Though some of the features of the inorganic chemical behavior of the metal may be retained, the bonding to the protein ligand usually alters many of them drastically (Williams, 1953). 

The metal is bound loosely to the protein and dissociates readily, and therefore the two cannot be isolated jointly in the natural state. Since the association is subject to variations in the physical-chemical environment, metal analyses of different preparations frequently yield varied and inconsistent results. The apoenzyme can be readily obtained metal-free, and the binding is therefore much less specific than that in metalloenzymes (Williams, 1953 Curd, 1954). 

It appears that four atoms of zinc are present in one molecule of ADH apoenzyme. The zinc in these crystalline preparations is firmly bound the zinc/protein bond is maintained against competitive physical-chemical factors involved in fractionation, potentially capable of dissociating it. Eecrystallization or dialysis against water fails to remove zinc. In some preparations more than 0.2 % of zinc was found. In these instances, small amounts of zinc could be removed by relatively mild procedures such as dialysis against OP. Such dialyses have not, thus far, lowered the zinc content below 4 moles of zinc per mole of protein. 

Reconstitution of apoproteins or apoenzymes with various synthetic hemes has been widely investigated in order to elucidate the effect of structural factors of the heme on the protein functions. If protoheme chemically modified at the 13- and 17-propionates forms a stable reconstituted... 

Coenzyme in the narrow sense, the dissociable, low-molecular-mass active group of an enzyme which transfers chemical groups (see Group transfer) or hydrogen or electrons. C. in this sense couple two otherwise independent reactions, and can thus be regarded as transport metabolites. In a wider sense, a C. can be regarded as any catalytically active, low-molecular-mass component of an enzyme. This definition includes C. that are covalently bound to enzymes as prosthetic groups. A holoenzyme consists of a C. in combination with an apoenzyme (enzyme protein). 

Vitamins function in two basic ways, either as a nutrient or vitamin or as a chemical (Herbert, 1980). When the function is known, fat-soluble vitamins function as regulators of specific metabolic activity, and the water-soluble vitamins function as coenzymes. Although rather exact roles for some of the vitamins in the chain of metabolic events are understood, it is safe to say that the complete function of any one of the vitamins in the body is unknown. What we do know about specific coenzyme functions is that the encouragement of doses of at least tenfold above the recommended dietary allowance (RDA) serves no nutritional function (Herbert, 1977). Vitamins enter the body as a component of food, travel to the tissues/cells that need them, are taken into the cells, and converted into a coenzyme form. In some cases the vitamin enters the cell as the coenzyme form already. A protein within the cell, called apoenzyme, combines with the vitamin coenzyme to form a holoenzyme. The holoenzyme or enzyme then serves the vitamin function of catalyzing certain specific metabolic-biochemical reactions. It appears that only when combined with its apoenzyme within the cell can a vitamin function as a vitamin. Since the quantity of protein, as well as the quantity of apoenzyme, any cell can make per unit time is limited (Schimke and Doyle,... 

Apoenzyme may be prepared from E. coli cells grown in the absence of added B12 compounds. Instability of the protein in this form has limited purification, but a partially purified preparation has been obtained (Weissbach et al., 1963) and, very recently, a means of chemical resolution of purified holoenzyme has been reported (Taylor, 1970). The apoenzyme is readily converted to holoenzyme by incubation with methyl-Bii in the absence of a reducing system (Guest et al., 1962 Weissbach et al., 1965). Ethyl-Bi2 and -propionate-Bi2 are also active. For cobalamins which possess no carbon-cobalt bond to react with apoenzyme to form holoenzyme a reducing system is required (Guest et al., 1960, 1962 Takeyama and Buchanan, 1961 Takeyama et al., 1961 ... 

The apoenzyme, the protein itself, has also been called a colloidal carrier. This terminology is based largely on Willstatter s idea that the molecule of an enzyme consists of a colloidal carrier and an active group with purely chemical activity. Today the concept of a colloidal carrier must be rejected, because it implies that the protein component is inactive, and we now know that it is not. For one thing, the protein component decides the substrate specificity, it determines which substances react and which do not. In many cases this same protein component also determines the direction of the reaction reaction specificity), in other words, which reaction out of the numerous possible ones is undergone by the substrate. This point becomes especially clear in cases where the same coenzyme, i.e. the same prosthetic group, catalyzes different reactions, as does, for instance, pyridoxal phosphate (see Chapt. VIII-4) or heme (see Chapt. IX-3). 

Very often, enzymic reactions are assisted by coenzymes or prosthetic groups. The chemical structure of these oofactors is discussed in the following chapter. They are low molecular weight substances of much simpler structure than the high molecular weight proteins (apoenzymes). Some of the coenzymes, e.g. pyridoxal phosphate, by themselves, without the apoenzyme, exhibit catalytic properties which can be explained by modem electron theory (cf. Chapt. VIII-4). Such explanations, however, do not answer the question why enzymic reactions occur so much faster and why they are guided in a very specific direction. The apoenzyme, the protein component of the enzyme, evidently is responsible for the latter phenomena, but to interpret its role in the reaction is much more difficult, although a few concepts have been developed very recently. 

Certain classes of enzymes require small, auxiliary, nonprotein molecules called cofactors, coenzymes, and prosthetic groups. Definitions for these three terms are somewhat arbitrary and, in fact, the term cofactor will be used in the following chapters to represent broadly the identity and functional roles of cocatalysts. The roles of cofactors are structural, functional, or both. They provide the enzyme with the chemical or photochemical capabilities lacking in the normal amino acid side chains. An enzyme devoid of a cofactor is called an apoenzyme. Apoenzymes are catalytically inactive. The active complex of the protein and the cofactor is termed a holoenzyme. The cocatalysts can be defined on the basis of the catalytic functions that are mediated (76). 

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