Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.

Cytochrome b5 binds spontaneously to lipid vescles and also self-associates in aqueous solution. Two mutant proteins have been generated, one has a self-association constant which is less than that of the native protein, while the other has a larger self-association constant. All three proteins have Trp in the membrane-binding domain but as aqueous solutions of these proteins contain differing amounts of monomeric protein, the kinetics of fluorescence enhancement, when the proteins are mixed with lipid vesicles, are complex. Similar complex kinetics are seen when the Trp are quenched by the addition of bromolipid vesicles. The mutant which has Trp 108 and 112 both replaced by Leu does not self-associate and shows monoexponential stopped-flow fluorescence kinetics. Identical rate constants are seen with this mutant for fluorescence enhancement by POPC and fluorescence quenching by three bromolipids with bromines at the 6,7-, 9,10-, and 11,12-positions of thesn-2 acyl chain. This rate constant is only 1% of the calculated collisional rate constant and it is suggested that the reduced rate is caused by a reduction in the number of productive collisions rather than by a slow rate of penetration of the membrane-binding domain into the bilayer.

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Purine dimers are formed by oxidation of DNA. There is evidence that these dimers are not repaired by cells from the human disease xeroderma pigmentosum. It has been suggested that unrepaired purine dimers are involved in the etiogenesis of internal cancers and neural degeneration that are observed in this disease. In order to study the properties and biological consequences of such moieties, these compounds were synthesized: 8-8-(2'-deoxyadenosyl)-2'-deoxyadenosine; 8-8-(2'-deoxyadenosyl)-2'-deoxyadenosine-5'-monophosphate; 8-8-(2'-deoxyadenosyl)-2'-deoxyguanosine; 8-8-(2'-deoxyadenosyl)-2'-deoxyguanosine-5'-monophosphate; 8-8-(2'-deoxyguanosyl)-2'-deoxyguanosine; 8-8-(2'-deoxyguanosyl)-2'-deoxyguanosine-5'-monophosphate; 8-8-(2'-deoxyguanosyl)-2'-deoxyadenosine, and 8-8-(2'-deoxyguanosyl)-2'-deoxyadenosine-5'-monophosphate. Following purification, they were characterized by mass spectrometry and nuclear magnetic resonance studies. Ultraviolet, fluorescence, and circular dichroic spectra of these products were established. The behavior of these photoproducts in various chromatographic systems was elucidated. Syntheses of purine dimers and descriptions of their properties can aid the studies of their possible formation in, and excision from, oxidized DNA.