Rates of Biotransformations Enzymes

Now we consider situations in which transformation of the organic compound of interest does not cause growth of the microbial population. This may apply in many engineered laboratory and field situations (e.g., Semprini, 1997 Kim and Hao, 1999 Rittmann and McCarty, 2001). The rate of chemical removal in such cases may be controlled by the speed with which an enzyme catalyzes the chemical s structural change (e.g., steps 2, 3 and 4 in Fig. 17.1). This situation has been referred to as co-metabolism, when the relevant enzyme, intended to catalyze transformations of natural substances, also catalyzes the degradation of xenobiotic compounds due to its imperfect substrate specificity (Horvath, 1972 Alexander, 1981). Although the term, co-metabolism, may be used too broadly (Wackett, 1996), in this section we only consider instances in which enzyme-compound interactions limit the overall substrate s removal. Since enzyme-mediated kinetics were characterized long ago by Michaelis and Menten (Nelson and Cox, 2000), we will refer to such situations as Michaelis-Menten cases. 

In general, Michaelis and Menten envisioned enzyme-mediated reactions as involving the following simple sequence  

In this conceptualization, it is assumed that the product is rapidly released and removed so that back reactions do not occur. With this somewhat simplified view (i.e., compare this sequence with somewhat more detailed enzymatic processes examined in Section 17.3), a kinetics expression for the removal of i can be written assuming the reaction step is rate limiting (see Box 12.2 for derivation)  

KiMM is given the subscript, MM, to remind us that it reflects Michaelis-Menten enzyme kinetics as distinguished from KiM used above to model microbial growth kinetics (see Monod cases above). Note, is the same as KE in Box 12.2 when it s value represents the reciprocal of the equilibrium constant for the binding step. 

Commonly, d [i /dt is referred to as the velocity of the reaction and denoted v. This reaction velocity expression captures the dependency of 6[i]ldt on [z] (see Fig. 17.16). Eq. 17-80 implies that the reaction velocity increases linearly with [z] (slope of kn [Enz]tot / KiMM) as long as [z] K,MM, and the reaction velocity is maximal (called Vmax = kE [Enz]Iot) when [z] is much greater than KiMM. Hence, Eq. 17-79 is commonly written  

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Attempts have been made to apply the structure-activity concept (Hansch and Leo 1995) to environmental problems, and this has been successfully applied to the rates of hydrolysis of carbamate pesticides (Wolfe et al. 1978), and of esters of chlorinated carboxylic acids (Paris et al. 1984). This has been extended to correlating rates of biotransformation with the structure of the substrates and has been illustrated with a number of single-stage reactions. Clearly, this approach can be refined with the increased understanding of the structure and function of the relevant degradative enzymes. Some examples illustrate the application of this procedure ... 

The individual reactions of drug metabolism will proceed at different rates depending upon the substrate (drug or metabolite), the enzyme catalyzing the particular reaction, and any other interfering agents. If the overall rate of biotransformation is slow, then the effects being observed are those primarily of the parent compound itself and perhaps, to a limited extent, to one or more of the initial metabolites. When the overall rate is fast, the effects must be due to the metabolite(s). 

Apart from the site and route of administration, formulation, dosage, and duration of treatment, biotransformation is often also affected by several other factors including age, species differences, sex differences, diet, diseases, hormones, and environment. The activity of the liver microsomal enzymes is low in newborns and aging animals resulting in a slower rate of biotransformation. Species differences in dosage and response are often due to biotransformation differences. Inadequate protein intake approaching starvation may also decrease the rate of biotransformation (12). Diseases of the liver sometimes also interfere with the normal biotransformation capacity. In addition, increase in biotransformation may occur at high body temperatures because of an increase in the metabolic rate. 

In sum, biotransformations may be limited by (1) delivery of the chemical to the organisms metabolic apparatus capable of transforming the chemical, (2) the enzyme s ability to mediate the initial transformation of the chemical, or (3) the growth of a population of microorganisms in response to the presence of a new substrate. Depending on what limits the rate of biotransformation, different mathematical frameworks are required to describe the kinetics of the process both with respect to the nature of the equations and the parameters they require. 

Finally, assuming bioavailability and biouptake do not limit the rate of biodegradation, then we expect the biodegradation kinetics to reflect the rate of growth due to utilization of a substrate or the rate of enzyme processing of that compound (both discussed in more details below). In these cases, the rate of biotransformation, khm, has been studied as a function of the substrate s concentration in the aqueous media in which the microorganisms or enzymes occur. Hence, for an environmental system, we can write ... 

Hence, sometimes phenomena associated with enzyme kinetics control the rate of biotransformations. If suitable enzymes are present in the microbial community, for example due to consumption of structurally related growth substrates, then we may see immediate degradation of compounds of interest like BQ when they are added to these metabolically competent microbial communities (Fig. 17.17). For such cases, if the abundance of the bacteria is varied, the rate of removal changes accordingly. Consequently, the removal of BQ could be described by a second-order rate law (Smith et al., 1978) ... 

The rate of biotransformation of a chemical depends on the amount and efficiency of the pertinent biotransformation enzymes. Enzyme activity is partly genetically determined but may also vary between and within people because of enzyme induction caused by previous exposure to the same or related chemicals. Variation in enzyme activity may also be caused by enzyme inhibition due to concurrent exposures. 

In an attempt to correlate pharmacological activity with the rate of biotransformation, Beckett and Morton have studied the metabolism of a series of indole alkaloids and related model systems by a variety of enzyme preparations of mammalian origin (39, 40, 57-59). Of the systems studied, rabbit liver microsomes were effective in performing O-demethylation liver microsome preparations from rat and guinea pig were not very effective in transforming alkaloids 4-7 and 9-19 (but vide infra) (40). In the case of corynantheidine (4), the product of metabolism was identified as the 0-( 17)-demethyl compound 8 by TLC comparison with authentic material. 

Enzymes are excellent catalysts in biological systems. They produce biologically active molecules by lowering the activation energy, thus causing the rate of biotransformation in the reaction to increase by several orders of magnitude. The general scheme for enzyme-catalyzed reactions is ... 

There are variations in the structures of these enzymes among individuals which can produce different rates of biotransformation of xenobiotics—hence, some people are more snsceptible to some toxicants while other people are more resistant to those toxicants. This may be because some people lack key enzymes, or, if present, these enzymes do not work well. This is why, for example, medications work well for some people but not others. 

The Arrhenius relation will not be observed above the temperature at which the decomposition or, as may occur for enzymes, inactivation of one or more of the reactants occurs (r ax)- Indeed, adherence to this relation at temperatures well above T ax for niost microorganisms has been used as evidence for an abiotic, rather than a biologically mediated mechanism of transformation (Wolfe and Macalady, 1992). For biotransformations, the Arrhenius equation also fails to describe the temperature dependence of reaction rates below the temperature at which biological functions are inhibited (T in), and above the temperature of maximum transformation rate (Topt). An empirical equation introduced by O Neill (1968) may be used to estimate the rates of biotransformation as a function of ambient temperature, min opt max (in this case, the lethal temperature), and the maximum biotransformation rate (p-max)- Because of the complexity of biochemical systems and the myriad of different structures encompassed by pesticide compounds, Tmiii, Topt, Tmax and p max are all likely to vary among different compounds, microbial species and geochemical settings (e.g., Gan et al., 1999, 2000). However, Vink et al. (1994) demonstrated the successful application of the O Neill function to describe the temperature dependence of biotransformation for 1,3-dichloro-propene and 2,4-D in soils (Figure 13). 

Water is known to be essential for the enzyme activity. Small amounts of water enhance enzyme activity, however excess water hinders the rate of some enzyme catalyzed reactions. Also, supercritical water cannot be used as the reaction medium either, because its critical temperature and pressure are too high for the enzymes used in biotransformations. The active site concentration on enzymes, hence the enzyme activity is found to be higher in the presence of hydrophobic supercritical fluids (ethane, ethylene) as compared to hydrophilic supercritical carbon dioxide. 

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