RATIONAL APPROACH TO IMPROVING REDUCTIVE CATALYSIS BY CYTOCHROME P450CAM

Although halogenated hydrocarbons are noted for low chemical reactivit y, small amounts are toxic to humans. Cytochromes P450 have been impli cated in transforming these compounds to more reactive species, under anaerobic conditions, through reduction at the heme. A significant amo unt of effort has been directed toward turning this catalytic ability to our advantage by engineering P450 variants than can efficiently rem ediate these compounds in situ, before they come in contact with the h uman population. We have taken a 'rational' approach to this problem, in which a combination of theory and molecular modeling is applied to identify which properties of the enzyme have the greatest influence ov er reductive dehalogenation. Recent progress in this area is briefly r eviewed. Two novel mutants, incorporating tryptophan (positions 87 and 396) and histidine (position 96, neutral and protonated) amino acid s ubstitutions in the active site, are proposed and evaluated using mole cular dynamics simulations. The upper bound on rate enhancement relati ve to wild-type is estimated in each mutant using electron transfer th eory. The most significant rate enhancement is predicted for the His 9 6 mutant in the protonated state; while some His residues of certain p roteins exhibit a pK(a) high enough to support a large protonated popu lation, such information is not presently available for this proposed mutant.