Dellar, S.; (2004) Comparison of the action of primary alcohols as inhibitors of a soluble and a membrane-bound cytochrome P450 hydroxylase. Doctoral thesis , University of London.

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Abstract

This thesis describes the development of a procedure for comparing the manner in which organic solvents inhibit biocatalysts. The research compared the effect of a series of primary alcohols on a membrane bound and a soluble cytochrome P450 hydroxylase. The effect of alkanes and primary alcohols on the bioluminescence of luciferase (EC 1.13.12.7) was developed as the basis of a fast and stable bioassay for measuring the aqueous concentration of a primary alcohol or an alkane (Franks and Lieb, 1984). This was then used to determine the concentration of organic solvents in the aqueous phase of a mixture of biological membranes and water. The bioassay was used in a study of the effect of primary alcohols on two cytochrome P450 hydroxylases. The first was a purified and soluble cytochrome P450, derived from Bacillus megaterium (recombinant P450 BM3 engineered for high expression in Escherichia coli (kindly supplied by Dr Munro at the University of Edinburgh)) and the second a membrane associated enzyme present in Rhizopus stolonifer (formally known as R. nigricans). The effect of the solvents in the reaction mixture was characterised by their IC50 value, that is, the concentration of the solvent that caused a 50 % inhibition of the enzyme activity. The soluble enzyme from B. megaterium was found to be more strongly inhibited by short chain alcohols (IC50 for methanol = 0.71 M) than the enzyme from R. stolonifer (IC50 for methanol =1.63 M). The inhibition of both enzymes by the primary alcohols becomes more extreme as their molecular weight increased, and they were almost equally inhibited by pentanol (IC50 =62 mM and 46 mM respectively). Hexanol inhibited the enzyme from R. nigricans (IC50 =25 mM) but was not sufficiently soluble in water (solubility limit is 50.1 M at 25 C,Bell, 1972) to inhibit the enzyme from B. megaterium. The aqueous concentration of various primary alcohols and alkanes in the presence of the cells of R. stolonifer suggested that at 50 % inhibition the molar concentration of the solvent in the membranes of the cells was the same for all the alcohols studied. The log IC50 (the logarithm of the concentration of the alcohols that caused a 50 % inhibition of the enzyme activity) was found to be a linear function of the carbon chain length of the alcohol and decreased regularly as the chain length increased. A multiple regression analysis showed that the effect of the series of primary alcohols on the two hydroxylases studies was significantly different.

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