Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase, from Saccharomyces cerevisiae.
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. 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. 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 situ from 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. The spin state of purified yeast cytochrome P-448 was 94% low spin (22 degrees 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 spin at 22 degrees C). 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 beta-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites. 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. 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.(ABSTRACT TRUNCATED AT 400 WORDS)
D000596:Amino Acids / Q000032:analysis; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001579:Benzopyrene Hydroxylase / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D019388:Cytochrome P-450 CYP1A2; D003580:Cytochromes / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000032:analysis; D007700:Kinetics; D010743:Phospholipids / Q000032:analysis; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D003580:Cytochromes; D010743:Phospholipids; D006418:Heme; D001579:Benzopyrene Hydroxylase; D001189:Aryl Hydrocarbon Hydroxylases; C017170:Cyp1a2 protein, rat; D019388:Cytochrome P-450 CYP1A2