Cosubstrates and cofactors

The Krebs cycle is presented in three figures. The first (Figure 4.57) depicts the cosubstrates and cofactors used in each reaction. The second (Figure 4.5S) gives names for the intermediates. The third (Figure 4.59) lists the names of the enzymes. 

Calculate the overall Kiq and AG at pH 7 and 25 C for the conversion of fumaric acid to citric acid in the presence of the appropriate enzymes, cosubstrates, and cofactors. 

The use of water-miscible organic solvent-water mixtures is a particularly attractive method for use with cofactor-dependent enzymes due to its simphcity. The high water content can allow dissolution of both enzyme and cofactor, whilst the water-miscible solvent can provide a dual role in both substrate dissolution and as a cosubstrate for cofactor recycling (substrate-coupled cofactor recycling).The asymmetric reduction of a ketone intermediate of montelukast using an engineered ADH in the presence of 50 % v/v isopropanol offers a powerful demonstration of this methodology (Scheme 1.55). 

One of our goals for studying TauD was to correlate our spectroscopic results with the structural information available from the X ray crystal structure of the ternary complex prepared under anaerobic conditions. Subsequently, this information could be used as a foundation to study similarities in catalytic mechanisms between different members of this enzyme class. Because the catalytic mechanism involves the displacement of bound water molecules from the Fe(II) cofactor, we studied samples of Fe(II)NO-TauD without cosubstrates and with just the aKG added to determine if FIYSCORE could be used to follow this chemistry. Figure 16(a) shows the FIYSCORE spectrum at g = 4.00 for Fe(II)NO TauD in aqueous buffer without the two cosubstrates. Two water molecules should be coordinated to the Fe(II)NO center under these conditions. The HYSCORE shows a weak, disordered system of overlapping H arcs for this sample, which most likely reflects a high degree of... 

Figure 29.6 Pathways for the metabolism of homocysteine. Normal transsulfuration requires cystathionine P-synthase with vitamin Bg as cofactor. Reme-thylation requires 5,10-methylenetetrahydrofolate reductase and methionine synthase. The latter requires folate as cosubstrate and vitamin Bi2 (cobalamin) as cofactor. An alternative remethylation pathway also exists using the cobalamin independent betaine-homocysteine methyltransferase (Robinson 2000).

Brain tryptophan-S-hydroxylase is a monooxygenase requiring molecular oxygen as a cosubstrate and an external electron donor, tetrahydrobiopterin, the cofactor also required by phenylalanine and tyrosine hydroxylases. 

Isopropanol can be used in the asymmetric hydrogen transfer reactions for two purposes first, as a cosolvent for improving the substrate solubility and second, as a cosubstrate for cofactor regeneration (substrate-coupled approach). For example, in the reduction of acetophenone by Candida viswanathii cells, addition of 10% (v/v) isopropanol led to a great increase of the substrate tolerance and a conversion of 90% compared to 9% in the control at a substrate concentration of 70 mM after lh[8]. 

All NOS isoforms utilize L-arginine as the substrate, and molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as cosubstrates. Flavin adenine dinucleotide (FMN), flavin mononucleotide (FAD), and (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) are cofactors of the enzyme. All NOS isoforms contain heme and bind calmodulin. In nNOS and eNOS,... 

Biochemical Effects Several enzymes that use nicotinamide cofactors were found to ye inhibited by PAN (at 125 ppm for 1 min) in in vitro studies. These enzymes were most susceptible in the absence of substrates. In some cases, an enzyme was protected by the nicotinamide cofactor (e.g., G-6-PD plus NADP), and in other cases, by the cosubstrate (e.g., isocitrate dehydrogenase plus isocitrate). Precisely the same protection could be obtained when compounds that react with sulfhydryl compounds (e.g., p-mercuricbenzoate) were used instead of PAN. Thus, the evidence indicated that PAN reacted with sulfhydryl groups. 

Cofactor regeneration, an economically essential step in the synthetic use of ADH, was accomplished within the PVA matrix using isopropanol as cosubstrate for the ADH itself or for a second alcohol dehydrogenase from Thermoanaerobium hrockii (E.C. 1.1.1.2). An overall turnover number of 10 was achieved, which is a promising magnitude for technical application. However, while the presence of the cosubstrate in the gel-stabilized two-phase system improved the solubility of substrates in the gel phase and consequently enhanced the productivity of the... 

Two classes of monooxygenases are known. Those requiring a cosubstrate (BH2 of Eq. 18-36) in addition to the substrate to be hydroxylated are known as external monooxygenases. In the other group, the internal monooxygenases, some portion of the substrate being hydroxylated also serves as the cosubstrate. Many internal monooxygenases contain flavin cofactors and are devoid of metal ions. 

The process of assaying for a particular reaction during purification may be complicated in cases requiring cosubstrates, coenzymes, or cofactors. Usually these additional requirements are discovered by the trial-and-error procedure of adding test substances to the reaction mixture and observing whether they accelerate the reaction or lead... 

Over the last 20 years, many reservations with respect to biocatalysis have been voiced, contending that (i) enzymes only feature limited substrate specificity (ii) there is only limited availability of enzymes (iii) only a limited number of enzymes exist (iv) protein catalyst stability is limited (v) enzyme reactions are saddled with limited space-time yield and (vi) enzymes require complicated cosubstrates such as cofactors. 

Because xenobiotic metabolism involves many enzymes with different cofactor requirements, prosthetic groups, or endogenous cosubstrates, it is apparent that many different nutrients are involved in their function and maintenance. Determination of the effects of deficiencies, however, is more complex because reductions in activity of any particular enzyme will be effective only if it affects a change in a rate-limiting step in a process. In the case of multiple deficiencies, the nature of the rate-limiting step may change with time... 

Phase II Reactions. As with phase I reactions, phase II reactions usually depend on several enzymes with different cofactors and different prosthetic groups and, frequently, different endogenous cosubstrates. All of these many components can depend on nutritional requirements, including vitamins, minerals, amino acids, and others. Mercapturic acid formation can be cited to illustrate the principles involved. The formation of mercapturic acids starts with the formation of glutathione conjugates, reactions catalyzed by the glutathione -transferases. 

Success with resting cells acting on penicillin G allowed Cho et al. [14] to demonstrate the ring expansion of penicillin G. By increasing the concentration of the substrate (penicillin G), cosubstrate (a-ketoglutarate), the chief cofactor, (Fe2+), and cells, the conversion of penicillin G to DAOG was improved. Using such improved conditions, activity with cell-free extracts was also demonstrated on 14 other penicillins. 

A new subclass of enzymes that use SAM and an Fe4S4 center to generate a S -deoxyadenosyl radical is beginning to emerge. This subclass, designated as class III enzymes, is postulated to use SAM specifically as a cosubstrate rather than a cofactor as in the class I enzymes, or a cofactor generator as in the class II enzymes. The distinction is that in class III enzymes, SAM is stoichiometrically... 

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