Cytochrome P-450 comprises a large super family of hemoproteins that collectively catalyze the metabolism of literally thousands of different compounds, including steroids, fatty acids, drugs and carcinogens. Although the general sequence of events that occur during the action of these enzymes are understood and are common to all members of the family, many of the enzymes exhibit properties that rarely occur with other enzymes. For example 1) Many of the enzymes catalyze the oxidation of NADPH to form hydrogen peroxide and water in the absence of substrates. 2) A substrate may enhance the formation of hydrogen peroxide and water. 3) Some of the enzymes have very broad substrate specificities. 4) Some of the enzymes several metabolites from a single substrate. Studies of the deuterium isotope effects on the metabolism of substrates and the formation of products are useful in differentiating plausible mechanisms by which these unusual events occur. We have derived equations for isotope effects on various parameters, for different plausible mechanisms, and have used these equations to develop testable hypotheses which can be used to differentiate the mechanisms. Studies of the isotope effects on the metabolism of testosterone by cytochrome P-450 2A1 revealed that testosterone promotes the formation of hydrogen peroxide and water. They also showed all of the products are formed from a common intermediate and that (delta-6)-testosterone is formed by a double hydrogen abstraction mechanism in which the 6alpha-hydrogen is abstracted first and then the 7alpha-hydrogen.
