Uncoupling Enzyme Stability and Coenzyme Issues

As illustrated in Fig. 5.2, the cytochromes P450 are notorious for their tendency to undergo abortive catalytic cycles with the production of superoxide, peroxide, or water according to the point in the cycle at which the relevant oxy complex collapses. Obviously, the intracellular formation of peroxide and superoxide is undesirable and these radicals have potential to damage the P450 protein or heme macrocycle (leading to enzyme inactivation), or to escape the active site to react with other cellular molecules [130]. There are a number of potential reasons underlying these phenomena in various P450s. Following the first reductive step in the cycle, the ferrous oxy complex can decay to form superoxide if the second electron is not delivered efficiently to enable the progression of the cycle and the formation of more powerful oxidant species. The ferrous oxy complex is relatively short-lived in the P450cam enzyme, but is stabilized by the presence of substrate [131]. However, the complex has a much shorter lifetime in, for instance, P450BM-3 [77], and its instability is clearly also a major issue underlying the uncoupling of several mammalian isoforms [120].

Many bacterial isoforms (e.g. P450BM-3 and P450cam) appear to have an excellent mechanism of avoiding non-productive electron transfer from the redox partner in absence of substrate. This occurs by substrate-dependent desolvation of the heme iron and conversion of the heme iron to a high-spin species with a concomitant positive shift in heme iron redox potential [12, 13]. This switch triggers the electron transfer process to heme iron only in the presence of substrate. This type of regulation appears much rarer in mammalian P450s, and various isoforms are isolated in mixed-spin or substantially high-spin forms even in absence of substrate. Electron transfer to a substrate-free P450 will inevitably lead to eventual decay of the ferrous oxy complex unless substrate can bind in advance of either oxy complex decay or further reduction (which will generate even shorter lived oxy species) [120].

A further potential reason for the collapse of the ferrous oxy or later intermediates in the cycle is the inappropriate positioning of the substrate in the active site (i.e. substrate location too distant from the oxy complex to enable it to be attacked and oxygenated). In addition, if the substrate-like molecule presents a bond that cannot be productively attacked (i.e. is too strong), then abortive decay of the fer-ryl-oxo (or ferric hydroperoxy) form should occur [18]. Obviously, the collapse of the ferryl-oxo species (with water formation) is less deleterious than peroxide production via ferric hydroperoxy collapse; but both mechanisms result in nonproductive oxidation of NAD(P)H coenzyme in the cell. A common conception is that the mammalian drug-metabolizing P450s are designed to have broad substrate specificity, enabling oxygenation of a wide range of physiological and xenobiotic substrates. Such broad specificity may be achieved by large, flexible active sites (as is clearly seen in the case of the human CYP3A4 enzyme) and frequent uncoupling of electron transfer from substrate oxygenation may be a necessary evil associated with having a P450 system capable of dealing with a plethora of different organic molecules [132]. By contrast, many bacterial P450s have very well-coupled electron systems and far more restricted substrate selectivity (e.g. the P450cam system) [8]. The major bacterial P450s (particularly P450cam and P450BM-3) also have much higher turnover rates than eukaryotic counterparts, and it is thus no surprise that such systems have been most popular in rational engineering and forced evolution experiments to create catalytically efficient P450 systems with desirable substrate recognition properties.

Heal Yourself With Qi Gong

Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

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