Substrate-depleted system

Several properties of hepatic microsomal AHH activity were compared in control and DBA-pretreated male little skates as shown in Table I. Following treatment there was an approximately 35-fold increase in specific enzyme activity, as quantitated by fluorescence of the phenolic metabolites formed (3, 21). The pH optimum, which was fairly broad, and the concentration of benzo(a)-pyrene (0.06 mM) that had to be added to the incubation mixture to achieve maximum enzyme activity were the same for both control and induced skate hepatic microsomes. The shorter periods observed for linearity of product formation with microsomes from the induced skates is thought to be related to the much higher AHH activity present, and may be due to substrate depletion or the formation of products which are inhibitory (i.e., compete with the MFO system as they are substrates themselves). A similar explanation may be relevant for the loss of linear product formation at lower microsomal protein concentrations in the induced animals. 

Inhibitors of HA synthesis have valuable therapeutic potential in the prevention of cancer invasion and metastasis. A novel inhibitor of HA synthesis has been identified, 4-methylumbelliferone (4-MU). However, this inhibitor does not have a direct effect on the HAS enzymes. Rather, it is glucuronidation of the inhibitor that depletes UDP-glucuronyl transferase stocks, required for the formation of UDP-glucuronic acid. Thus, it is substrate depletion that appeeirs to be the mechanism of action. The inhibitor is effective in both vertebrate and bacterial systems [131]. 4-MU has been shown to inhibit liver metastases of melanoma following oral administration in mice [132]. 

Phenols, quantitatively important P450-derived metabolites of aromatic hydrocarbons, are substrates for both UDP-GT and sulfotransferases. Generally, glucuronide metabolites predominate after administration of a phenol or phenol precursor to mammals because sulfate formation is a high-affinity, low-capacity (due to sulfate depletion) system, whereas glucuronidation is a low-affinity, high-capacity system. 

Initial experiments involved the use of hydroquinones, ascorbate and dihydroxy fumarate as substrates for the enzyme [120], Radicals (e.g., 5 and 6) were detected under steady-state conditions using a flow system to minimize substrate depletion. The narrow line spectra (Fig. 6a,b) of the radical anions are identical to those generated in chemical systems, indicating that the radicals are free in solution rather than associated with the enzyme. If it is assumed that the reaction proceeds by one-electron oxidation of the substrate and that the product radicals decay by self-reaction (e.g., disproportionation), kinetic analysis predicts that the steady-state radical... 

Reactions of this kind have been postulated [161] to account for semiquinone production from the oxidation of catechol by tyrosinase and the reduction of p-benzoquinone and menadione by DT-diaphorase [159]. The rate of radical production is very low compared with the rate of substrate depletion, indicating that the free radical is not the major primary product of the enzyme reaction. As in the peroxidase system, substrate-derived radicals having an appropriate structure can be spin-stabilized with metal ions. 

Quantitation of metabolites is challenging in the absence of radiolabel or authentic standards. The most commonly used methods for metabolite monitoring are detection by ultraviolet (UV) absorbance and by mass spectrometry. When UV absorbance is being used for detection, the peak response will be approximately proportional to the amount of metabolite if the chromophore is not altered, such as hydroxylation of an aliphatic side group. However, if the molecule is cleaved, altering conjugated systems, relative UV peak responses may not accurately represent the relative amounts of parent compound and each metabolite. Mass spectrometry is even less reliable than UV absorbance in the absence of authentic standards, as ionization efficiency for each detected molecule will differ. In practice, a combination of UV and mass spectrometry is most appropriate. However, unlike the substrate depletion approach, the possibility would still exist of missing metabolites if the detection method does not pick up the metabolite of interest. 

Oxygen is used in these microbiolreactions to degrade substrates, in this case organic wastes, to produce energy required for ceU synthesis and for respiration. A minimum residual of 0.5 to 2.0 mg/L DO is usually maintained in the reactors to prevent oxygen depletion in the treatment systems. 

---