Rettie A E, Lawton M P, Sadeque A J, Meier G P, Philpot R M
Department of Medicinal Chemistry, University of Washington, Seattle 98195.
Arch Biochem Biophys. 1994 Jun;311(2):369-77. doi: 10.1006/abbi.1994.1250.
Multiple forms of the microsomal flavin-containing monooxygenase (FMO) exist in rabbit tissues. In order to better understand the catalytic properties of these isoforms, we have expressed rabbit FMO1, FMO2, FMO3, and FMO5 in Escherichia coli and examined their kinetic parameters and prochiral selectivities for the sulfoxidation of methyl-, ethyl-, n-propyl-, and n-butyl-substituted p-tolyl sulfides. FMO1 and FMO2 exhibited high affinities for these substrates (Km < 10 microM), in contrast to the low-affinity FMO3 form for which Km values ranged between 100 and 280 microM. FMO5 did not form quantifiable levels of sulfoxide metabolites at the concentrations used. The individual stereochemical metabolite profiles generated by FMO1, FMO2, and FMO3 were unique and served to distinguish among these three cDNA-expressed isoforms. To investigate the relationship between the kinetic parameters for the cDNA-expressed enzymes and the native microsomal enzymes, we examined the kinetics and stereoselectivity of metabolism of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes. We analyzed these data with respect to FMO1 and FMO3, the two predominant hepatic isoforms. Sulfoxidation of methyl p-tolyl sulfide by FMO1 and FMO3 solubilized from E. coli microsomes proceeded with apparent Kms of 18 and 270 microM, respectively. FMO1 was essentially stereospecific for formation of (R)-methyl p-tolyl sulfoxide, whereas FMO3 generated this metabolite with little prochiral selectivity. Sulfoxidation of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes was best described by a two-enzyme model, with apparent Km values of 11 and 340 microM. The enantiomeric purity of the (R)-methyl p-tolyl sulfoxide metabolite, generated by detergent-solubilized rabbit liver microsomes, decreased progressively with increasing substrate concentration, from a high of 96% enantiomeric excess at a substrate concentration of 5 microM to a low of 63% enantiomeric excess at a substrate concentration of 2 mM. The kinetic and stereochemical properties of the high-affinity and low-affinity components of detergent-solubilized rabbit liver microsomes were similar to those exhibited by cDNA-expressed FMO1 and FMO3, respectively. Therefore, methyl p-tolyl sulfide, used at the appropriate substrate concentrations, is useful for discriminating between FMO1- and FMO3-mediated catalysis in rabbit liver microsomal preparations.
兔组织中存在多种形式的微粒体含黄素单加氧酶（FMO）。为了更好地了解这些同工型的催化特性，我们在大肠杆菌中表达了兔FMO1、FMO2、FMO3和FMO5，并检测了它们对甲基、乙基、正丙基和正丁基取代的对甲苯基硫醚进行亚砜化反应的动力学参数和前手性选择性。FMO1和FMO2对这些底物表现出高亲和力（Km<10μM），而低亲和力的FMO3形式的Km值在100至280μM之间。在所使用的浓度下，FMO5未形成可定量水平的亚砜代谢物。FMO1、FMO2和FMO3产生的各个立体化学代谢物谱是独特的，可用于区分这三种cDNA表达的同工型。为了研究cDNA表达的酶与天然微粒体酶的动力学参数之间的关系，我们检测了去污剂增溶的兔肝微粒体对甲基对甲苯基硫醚的代谢动力学和立体选择性。我们针对两种主要的肝脏同工型FMO1和FMO3分析了这些数据。从大肠杆菌微粒体中增溶的FMO1和FMO3对甲基对甲苯基硫醚的亚砜化反应的表观Km分别为18和270μM。FMO1对（R）-甲基对甲苯基亚砜的形成基本上具有立体特异性，而FMO3生成这种代谢物时前手性选择性很小。去污剂增溶的兔肝微粒体对甲基对甲苯基硫醚的亚砜化反应最好用双酶模型来描述，表观Km值为11和340μM。去污剂增溶的兔肝微粒体产生的（R）-甲基对甲苯基亚砜代谢物的对映体纯度随着底物浓度的增加而逐渐降低，从底物浓度为5μM时的96%对映体过量的高值降至底物浓度为2mM时的63%对映体过量的低值。去污剂增溶的兔肝微粒体的高亲和力和低亲和力组分的动力学和立体化学性质分别与cDNA表达的FMO1和FMO3所表现出的性质相似。因此，在适当的底物浓度下使用甲基对甲苯基硫醚，可用于区分兔肝微粒体制剂中FMO1和FMO3介导的催化作用。
