Suh J K, Poulsen L L, Ziegler D M, Robertus J D
Department of Chemistry, University of Texas, Austin, Texas, 78712, USA.
Arch Biochem Biophys. 1999 Dec 15;372(2):360-6. doi: 10.1006/abbi.1999.1530.
The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.
来自酿酒酵母的含黄素单加氧酶（yFMO）利用NADPH和O₂氧化含硫醇的底物，如谷胱甘肽（GSH），从而为内质网产生氧化电位。该酶利用NADPH的效率比NADH高12倍。氨基酸序列分析表明，赖氨酸219和/或赖氨酸227可能作为NADPH 2'磷酸基团的抗衡离子，并有助于确定对吡啶核苷酸的偏好。定点突变表明，赖氨酸219对共底物识别的贡献更大。将赖氨酸219突变为丙氨酸会使依赖NADPH的活性降低90倍，但对依赖NADH的活性没有影响。将赖氨酸227突变为丙氨酸会使依赖NADPH的活性降低五倍，使依赖NADH的活性降低三倍。通过光谱法测量了NADP⁺与氧化型yFMO的解离常数。野生型酶的Kd为12 μM，K219A突变体的Kd为243 μM，这与赖氨酸219在吡啶核苷酸结合中的作用一致。
