Cofactors commonly encountered

Cofactors Commonly Encountered in Biocatalytic Reactions Applied to Chemical Synthesis... 

Some enzymes associate with a nonprotein cofactor that is needed for enzymic activity. Commonly encountered cofactors include metal ions such as Zn2+ or Fe2+, and organic molecules, known as coenzymes, that are often derivatives of vitamins. For example, the coenzyme NAD+contains niacin, FAD contains riboflavin, and coenzyme A contains pantothenic acid. (See pp. 371-379 for the role of vitamins as precursors of coenzymes.) Holoenzyme refers to the enzyme with its cofactor. Apoenzyme refers to the protein portion of the holoenzyme. In the absence of the appropriate cofactor, the apoenzyme typically does not show biologic activity. A prosthetic group is a tightly bound coenzyme that does not dissociate from the enzyme (for example, the biotin bound to carboxylases, see p. 379). 

The electrons that must be delivered to the heme iron to accomplish catalysis are transferred from the reduced cofactor NAD PH by the redox partners. Nature uses a variety of strategies to accomplish this, almost all of which rely on intermediary electron transfer proteins that themselves require nonprotdnaceous cofactors. There are currendy at least 10 different systems or classes known [15], but for the purposes of applied biocatalysis, it is important to be aware of the four most commonly encountered in the biocatalysis literature (Figure 8.4). 

The reactions known to occur in the denitrification and nitrification pathways are summarized in Table 1, along with the most commonly encountered cofactors. 

Disorders in the metabolism of propionyl-CoA and methylmalonyl-CoA have been recorded at every step of these pathways and are further augmented by disorders in the synthesis of the 5 -deoxyadenosylcobalamin cofactor required by methylmalonyl-CoA mutase and of propionyl-CoA holo-carboxylase due to deficient activity of holocarboxylase synthetase (Section 10.3.2). Collectively these disorders comprise probably the most commonly encountered abnormal organic acidurias and this chapter describes these diseases and their underlying biochemistry. 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

Another way of bringing reactants into close proximity, which is encountered commonly in transition metal chemistry, is through metal ion complexation. The coordination of a reactant to a metal ion complex often activates its reactivity and can bring the reactant into close proximity with a second reactant or with a catalytic group. One example, shown in Fig. 6, is a zinc (11) complex of 1,5,9-triazacyclononane, as a model for the enzyme carbonic anhydrase, which contains a zinc (11) cofactor in its active site (4). In the aqua complex, the bound water molecule has a dramatically reduced pKa value of 7.3, which is similar to the pKa of the active site nucleophihc water. The corresponding cobalt (111) complex catalyzed ester hydrolysis at twice the rate because Co(lll) can coordinate both the hydroxide nucleophile and the ester carbonyl via a... 

Fig. 15 Protoporphyrin IX is a common cofactor in metalloproteins. When it incorporates iron, it is known as heme b. Further modifications to the macrocycle are encountered modification of the vinyl groups to secondary thiols gives heme c, which may be covalently incorporated into proteins via disulfide linkages...

The most common practice is to use an extinction coefficient of 1.0 when the purity and characteristics of the molecule are being determined. The sensitivity is typically in the range of 0.05 1.0 mg/mL, and one must be aware of the fact that impurities, particularly those that contain aromatic side chains, may lead to inaccuracies. Further interference that can skew results may be encountered with pigments, organic cofactors, and phenolic constituents of the solution. In addition, the extinction coefficient for a protein is pH dependent. On the whole, this approach is the most common one in use and although it can only be expected to yield an estimate of the true concentration, sometimes this is the best that can be done. 

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