Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK...Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu^370 which is crucial for many hydroxylations.展开更多
The binding between NL-101, a novel nitrogen mustard anti-cancer drug, with amino acids and peptides has been investigated by high performance liquid chromatography electrospray tandem mass spectrometry(HPLC/ESI-MS/MS...The binding between NL-101, a novel nitrogen mustard anti-cancer drug, with amino acids and peptides has been investigated by high performance liquid chromatography electrospray tandem mass spectrometry(HPLC/ESI-MS/MS). This study offers supporting data of the interaction among drug and amino acids and peptides, which could potentially explain the cytotoxic and mutagenic effects of the drug. Collision-induced dissociation(CID) experiment demonstrated that under the same collision energy, the amino group combined with NL-101 adducts are sensitive and often produce more fragment ions; the carboxyl group combined with NL-101 adducts are hard to break and display fewer fragment ions. In addition, when other group(like sulfhydryl group) of amino acids binds to NL-101, CID spectra show different fragmentation pattern. These differences could display structural information about the drug adducts and be utilized as location of the authentic binding sites.展开更多
文摘Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu^370 which is crucial for many hydroxylations.
基金supported by the National Natural Science Foundation of China (Nos. 21327010, 21372199)
文摘The binding between NL-101, a novel nitrogen mustard anti-cancer drug, with amino acids and peptides has been investigated by high performance liquid chromatography electrospray tandem mass spectrometry(HPLC/ESI-MS/MS). This study offers supporting data of the interaction among drug and amino acids and peptides, which could potentially explain the cytotoxic and mutagenic effects of the drug. Collision-induced dissociation(CID) experiment demonstrated that under the same collision energy, the amino group combined with NL-101 adducts are sensitive and often produce more fragment ions; the carboxyl group combined with NL-101 adducts are hard to break and display fewer fragment ions. In addition, when other group(like sulfhydryl group) of amino acids binds to NL-101, CID spectra show different fragmentation pattern. These differences could display structural information about the drug adducts and be utilized as location of the authentic binding sites.
