Cofactor consumption

The overall reaction rate not only determines the necessary reaction time to reach the desired conversion (see Fig. 7-23) but also the enzyme and cofactor consumption, both being strongly influenced by the reactor conditions. 

Cofactor consumption is specified with the total turnover number , defined as mols cofactor consumed per mol of product formed. 

In oiological systems, the most frequent mechanism of oxidation is the remov of hydrogen, and conversely, the addition of hydrogen is the common method of reduc tion. Nicotinamide-adenine dinucleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP) are two coenzymes that assist in oxidation and reduction. These cofactors can shuttle between biochemical reac tions so that one drives another, or their oxidation can be coupled to the formation of ATP. However, stepwise release or consumption of energy requires driving forces and losses at each step such that overall efficiency suffers. 

In a first reactor, where benzoylformate decarboxylase (BFD) is retained, benz-aldehyde and acetaldehyde are coupled to yield (S)-hydroxy-l-phenylpropanone. This hydroxy ketone is then reduced to the corresponding diol in a second reactor by an alcohol dehydrogenase (ADH). Regeneration of the necessary cofactor is achieved by formate dehydrogenase (FDH) or by other methods. To avoid additional consumption of redox equivalents by unselective reduction of residual starting material from the first reactor, the volatile aldehydes are removed via an inline stripping module between the two membrane reactors. In this setup the diol was produced with high optical purity (ee, de > 90%) at an overall space-time yield of 32 g L d . ... 

This mechanism is consistent with a number of observations. Kinetic studies on prolyl 4-hydroxylase [223] and thymine hydroxylase (EC 1.14.11.6) [224] suggest that cofactor binds first, followed by 02. The bound 02 appears to have superoxide character, as superoxide scavengers are competitive inhibitors of 02 consumption [225,226], It is also clear that the oxidative decarboxylation of the keto acid is a distinct phase of the mechanism from the alkane functionalization step, as these two phases can be uncoupled, particularly when poor substrate analogs are employed [227-229], Evidence for an Fe(IV) = 0 intermediate derives from studies with substrate analogs. Besides the hydroxylation of the 5-methyl group of thymine, thymine hydroxylase can also catalyze ally lie hydrox-ylations, epoxidation of olefins, oxidation of sulfides to sulfoxides, and N-de-... 

The multilayer membrane coverage (of Fig. 6.6) improves the relative surface availability of oxygen and excludes potential interferences (common at the potentials used for detecting the peroxide product). Electrocatalytic transducers based on Prussian Blue layers (15) or metallized carbons (16), which preferentially accelerate the oxidation of hydrogen peroxide, are also useful for minimizing potential interferences. The enzymatic reaction can also be followed by monitoring the consumption of the oxygen cofactor. 

NADPH balances are often essential for metabolite balancing based estimations of the net fluxes in a metabolic network. However, NADPH consumption and generation are often found in bidirectional reactions that cannot be quantified by metabolite balancing approaches. The mannitol cycle (Fig. 9) is an example of a pathway that can affect the NADPH balance, but has no net conversion of any metabolites, except for cofactors. In the mannitol cycle, NADH and NADP+ are converted into NAD+ and NADPH, respectively, at the expense of ATP [52]. Because mannitol happens to be symmetrical, the activity of the mannitol cycle will cause scrambling of the carbon atoms of fructose 6-phosphate, and the activity of the cycle may therefore be identified using labeling analysis. The mannitol cycle has been reported to be present in several fungi [52]. 

The in vitro oxidation of organic recalcitrant compounds by peroxidases requires that the enzyme, cofactors, and the compound to be degraded are all present in the reaction mixture [8, 22,127,128], However, one main drawback found is that there is an important consumption and destabilization of the enzyme in the process, which may limit the applicability of the enzymatic process. 

Either the CO2 formation is followed potemiometrically (243) or the O2 consumption is measured amperometrically at an oxygen electrode (245). In the first method, the enzyme is physically immobilized with a dialysis membrane. The response is linear in the range 5-300 pg/mL of salicylate. The second technique uses chemically immobilized enzyme (GA -F BSA) attached to a pig intestine mounted on the tip of the O 2 electrode. Samples containing from 10 pM to 2 mM salicylate were analyzed. An elegant microelectrode (244) has the enzyme and the cofactor immobilized in the electrode matrix (carbon paste) and the catechol formation is monitored at -F 300 mV versus Ag/AgCl. The electrode consists of a disposable strip, allowing measurements to be made on a drop of blood within 1 min. 

The basal activity of aminotransferase fell by 50% during consumption of the Bt-deficient diet. The stimulation occurring with addition of PLP to the enzyme incubation mixtures rose in this period from 200% (twofold stimulation) to 400% (fourfold). The basal activity of the aminotransferase, and probably of ail enzymes of the body, varies from subject to subject. It may even vary with repeated enzyme assays using the same sample of red blood cells. Thus, the basal activity is not used to assess vitamin Be, status. The percentage stimulation is relatively constant in normal subjects and is thus a more useful indicator of B status. It should be noted that the storaffe of ivti blood teiis can lead to gradual release of the cofactor from the enzyme thus, an artefactual diagnosis of Bh deficiency is possible. 

DHA and Coenzyme Q10. Coenzyme Q10 (CoQlO) is an essential cofactor involved in the mitochondrial electron transport chain. Zinc toxicity also affects cellular energy production by decreasing oxygen consumption rate (OCR) and ATP turnover in human neuronal cells, which can be restored by the neuroprotective effect of docosahexaenoic acid (DHA). DHA is specifically neuroprotective against zinc-triggered mitochondrial dysfunction, and CoQlO has shown to be protective against both Ap- and zinc-induced alterations in mitochondrial function [502],... 

The periplasmatic vanadium-containing nitrate reductase from P. isachenkovii has a molecular mass of 220 kDa (four subunits). The pterin cofactor is again absent. In media supplemented with vanadate and nitrate, vanadate is first reduced by a membrane-bound reductase using NADH as electron donor. This dissimilatory reduction was followed by nitrate consumption.I " ]... 

The cofactor of ADH, NAD+, may not be replaced by other electron acceptors. Malinauskas and Kulys (1978) attempted to construct a reagentless alcohol sensor by coimmobilizing ADH with dextran-bound NAD+ by a dialysis membrane in front of an oxygen electrode. The O2 consumption was indicated via the reoxidation of NADH by NMP+. The system has also been used to measure NAD+ with high sensitivity. In... 

Papirmeister and colleagues (1985) have followed another line of argument. They have shown that activation of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP) leads to the consumption of the cofactor NAD+. They argued that depletion of this cofactor explained the inhibition of glycolysis reported by Dixon and Needham (1946) and by Renshaw (1946). Other... 

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