Lightning L K, Jones J P, Friedberg T, Pritchard M P, Shou M, Rushmore T H, Trager W F
Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195, USA.
Biochemistry. 2000 Apr 18;39(15):4276-87. doi: 10.1021/bi992412u.
Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.
利用重组人细胞色素P450 3A4，对默克公司合成的潜在HIV蛋白酶抑制剂L-754,394基于机制的人肝P450 3A4失活作用进行了研究。酶失活的特征为分配系数较小（3.4或4.3±0.4），即抑制剂经历的代谢事件总数除以酶失活事件数；长时间透析后不可逆；相对于对照，450 nm特征峰无降低；脱辅基蛋白发生共价修饰。P450 3A4失活过程中形成的主要和次要产物分别是L-754,394的单羟基化和二氢二醇代谢物。与P450 3A4结合的L-754,394在用于SDS-PAGE、Ni(2+)亲和色谱和蛋白酶消化的条件下被水解。此外，该修饰对HPLC分离的酸性条件和CNBr消化不稳定。肽加合物的不稳定性质以及失活物质与P450 3A4的非化学计量结合，使得无法直接鉴定共价修饰的氨基酸残基或其连接的肽段。然而，Tricine SDS-PAGE结合MALDI-TOF-MS和同源性建模，初步确定I257-M317为活性位点肽段，而基于对活化呋喃的N-、O-和S-连接缀合物稳定性的先验知识，推测Glu307是被L-754,394标记的活性位点氨基酸。
