Iwasaki M, Juvonen R, Lindberg R, Negishi M
Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1991 Feb 25;266(6):3380-2.
The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively. This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase. We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s. The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form. Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms. This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s. This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.
在P450coh和P450(15)α中，第209位氨基酸的特性对于分别决定香豆素7-羟化酶和甾体15α-羟化酶的活性最为关键。因此，该系统为我们提供了一个绝佳的模型，用于研究作为单加氧酶的P450特异性的结构基础。我们在酿酒酵母中表达了一系列第209位残基被各种氨基酸取代的突变型P450，并对这些突变型P450的光谱特性和羟化酶活性进行了表征。在第209位存在包括苯丙氨酸、亮氨酸和缬氨酸在内的疏水残基会导致P450转变为高自旋状态，而带电荷的氨基酸如赖氨酸或天冬氨酸则产生低自旋形式。此外，该位置为天冬酰胺或甘氨酸的P450所呈现的光谱表明是高自旋和低自旋形式的混合。这种自旋改变取决于第209位残基的疏水性和大小，表明该位置可能位于这些P450血红素远端面第六个轴向配体附近。第209位残基与配体的这种接近性可能解释了该残基在决定这些P450的羟化酶特异性和活性方面的关键作用。
