Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase, from Saccharomyces cerevisiae

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Abstract
1. The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88–97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE. 2. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5–6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE. 3. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H2O2 generated in situfrom a glucose oxidase system; in each case Vmax is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased Km, is lowered. 4. The spin state of purified yeast cytochrome P-448 was 94% low spin (22°C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spinat 22°C). 5. Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from β-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites. 6. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine. 7. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources. 8. A more specific and efficient form of benzo[a]pyrene hydroxylase is induced by the addition of benzo[a]pyrene to the yeast growth medium at zero time. The efficiency of the enzyme, as indicated by the Vmax / Km ratio, increases progressively with concentration of benzo[a]pyrene. This indicates that multiple forms of yeast cytochrome P-448 occur. Induction of more efficient forms occurs at the expense of less efficient forms as little increase in total enzyme concn. is observed.

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