Apoenzymes

The catalytic subunit then catalyzes the direct transfer of the 7-phosphate of ATP (visible as small beads at the end of ATP) to its peptide substrate. Catalysis takes place in the cleft between the two domains. Mutual orientation and position of these two lobes can be classified as either closed or open, for a review of the structures and function see e.g. [36]. The presented structure shows a closed conformation. Both the apoenzyme and the binary complex of the porcine C-subunit with di-iodinated inhibitor peptide represent the crystal structure in an open conformation [37] resulting from an overall rotation of the small lobe relative to the large lobe. 

Figure 10. The G-protein cascades in smooth muscle catalyze the exchange GDP for GTP on G-protein. Following the binding of GTP, the trimeric G-protein splits into an a-GTP part and a P-y part. The a-GTP part ordinarily then combines with its specific apoenzyme to constitute the active enzyme. For the activation of the contractile activation path, the enzyme is phospholipase C and the second messenger products are IP3 and DAG. The IP3 in the myoplasm binds to Ca channels in the SR membrane, opening them. Other second messengers include the inhibitors of contractile activity, cGMP and cAMP.

Henn, SW Ackers, GK, Molecular Sieve Studies of Interacting Protein Systems. V. Association of Subunits of D-Amino Acid Oxidase Apoenzyme, Biochemistry 8, 3829, 1969. 

Flavoprotein enzymes contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed in the body from the vitamin riboflavin (Chapter 45). FMN and FAD are usually tighdy—but not covalendy—bound to their respecdve apoenzyme proteins. Metalloflavopro-teins contain one or more metals as essential cofactors. 

These dehydrogenases use nicotinamide adenine dinucleotide (NAD ) or nicotinamide adenine dinucleotide phosphate (NADP )—or both—and are formed in the body from the vitamin niacin (Chapter 45). The coenzymes are reduced by the specific substrate of the dehydrogenase and reoxidized by a suitable electron acceptor (Figure 11-4). They may freely and reversibly dissociate from their respective apoenzymes. 

Biotin functions to transfer carbon dioxide in a small number of carboxylation reactions. A holocarboxylase synthetase acts on a lysine residue of the apoenzymes of acetyl-CoA carboxylase, pymvate carboxylase, propi-onyl-CoA carboxylase, or methylcrotonyl-CoA carboxylase to react with free biotin to form the biocytin residue of the holoenzyme. The reactive intermediate is 1-7V-carboxybiocytin, formed from bicarbonate in an ATP-dependent reaction. The carboxyl group is then transferred to the substrate for carboxylation (Figure 21—1). 

D-Aminoacid oxidase has been isolated from a nnmber of yeasts, and the nucleotide sequence of the enzyme from Rhodotorula gracilis ATCC 26217 has been established (Alonso et al. 1998). The gene could be overexpressed in Escherichia coli, and levels of the enzyme were greater under conditions of low aeration the enzyme isolated from the recombinant organisms was apparently the apoenzyme since maximum activity required the presence of FAD. 

Lequea et al. used the activity of tyrosine apodecarboxylase to determine the concentration of the enzyme cofactor pyridoxal 5 -phosphate (vitamin B6). The inactive apoenzyme is converted to the active enzyme by pyridoxal 5 -phosphate. By keeping the cofactor the limiting reagent in the reaction by adding excess apoenzyme and substrate, the enzyme activity is a direct measure of cofactor concentration. The enzymatic reaction was followed by detecting tyramine formation by LCEC. The authors used this method to determine vitamin B6 concentrations in plasma samples. 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

Synthetic electron acceptors have been shown to react very rapidly with free flavins The combination of a flavin with an apoenzyme often inhibits the reaction with certain electron acceptors. The reaction with an electron acceptor will be thermodynamically favored if its standard reduction potential is larger than that of the flavin. A list of electron acceptors along with their reduction potentials can be foimd in Table 3. 

Spin labeled 5 -deoxyadenosylcobinamide has been used as a cofactor for ethanolamine-ammonia-lyase and the ESR spectrum followed during catalysis (123). This spin labeled coenzyme is biologically active in this enzyme. Enzyme kinetics showed this derivative to have the same Vmax as the cofactor 5 -deoxyadenosylcobinamide, but it has a higher Km value of 5.1 X 10-6 M compared to 4.6 X 10-6 for 5 -deoxyadenosylcobinamide (123). When the spin labeled coenzyme was incubated with apoenzyme to give the enzyme-coenzyme complex, the nitroxide ESR spectrum resembled that of free spin label but the lines are broadened significantly. 

Chinnayelka, S. and McShane, M. J. (2005). Microcapsule biosensors using competitive binding resonance energy transfer assays based on apoenzymes. Anal. Chem. 77, 5501-11. 

Enzymes may not function well or at all unless some other species known as a cofactor is present. An enzyme alone is referred to as the apoenzyme and the combination of enzyme and cofactor is known as the holoenzyme. Among the species that function as cofactors are organic compounds that interact with the enzyme. If the organic moiety is strongly attached to the enzyme, it is called a prosthetic group, but if it is loosely bound to the enzyme, it is referred to as a coenzyme. For the purposes of this discussion, the most interesting cofactors are metal ions. Depending on the type of enzyme, the appropriate metal ion cofactor may be Mg2+, Ca2+, K+, Fe2+, or Cu2+. A sizeable number of enzymes are sometimes called metalloenzymes because they have active sites that contain a metal. 

The enzyme mediating remethylation, 5-methyltetrahy-drofolate-betaine methyltransferase (Fig. 40-4 reaction 4), utilizes methylcobalamin as a cofactor. The kinetics of the reaction favor remethylation. Faulty remethylation can occur secondary to (1) dietary factors, e.g. vitamin B12 deficiency (2) a congenital absence of the apoenzyme (3) a congenital inability to convert folate or B12 to the methylated, metabolically active form (see below) or (4) the presence of a metabolic inhibitor, e.g. an antifolate agent that is used in an antineoplastic regimen. 

Sixteen patients had an associated hyperammonemia, citrullinemia and hyperlysinemia. This presentation is the most malignant, with death in early infancy. This French phenotype is commonly associated with the absence of any immunological cross-reacting material (CRM) corresponding to the pyruvate carboxylase apoenzyme protein. 

The inactive form GOin, which displays a typical Cu(II) EPR signal, yields upon one-electron oxidation the EPR silent active form GO0X. For many years the presence of a Cu(III) ion (ct,. S = 0) in the active site (121) of the fully oxidized state GO0X was assumed. The Whittakers (122) showed in 1990 that one-electron oxidation of the copper depleted apoenzyme of GO produced an EPR active, remarkably stable Tyr radical that was studied by UV-vis, EPR, and ENDOR spectroscopy. From these studies, they concluded that the thioether modified Tyr 272 was oxidized and, consequently, they proposed that GOcx contains a Tyr 272 radical coordinated to a Cu(II) ion. 

Coenzymes complement the catalytic action of the amino-acid functional groups. They are bound to apoenzymes (apoproteins) either covalently or non-covalently. It is interesting to note that non-covalently-bound coenzymes are polyanions at neutral pH as exemplified by the structures of glutathione (GSH) [17] and thiamine pyrophosphate [18]. Shinkai and Kunitake (1976b, 1977a) demonstrated the efficient binding of glutathione and coenzyme A (a polyphosphate) to cationic micelles and cationic polysoaps. Thus, the combina- ... 

An apoenzyme is the part of a conjugated en2yme without the prosthetic group. 

Pyridoxal Phosphate.—Analogues of pyridoxal and pyridoxamine 5 -phosphates have frequently been used to probe the size and shape of the active sites of a number of enzymes. For example, the apoenzyme of a tryptophanase from Bacillus alvei will bind pyridoxal 5 -phosphate as well as the 2-nor, 2 -methyl, 2 -hydroxy, 6-methyl, and A-oxide analogues.27 No analogue that has been modified at C-4 binds to the enzyme, confirming the absolute requirement for Schiff-base formation between the... 

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