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Objective: We have continued previous work in which we demonstrated that #117 and #372 amino acids contrib- uted to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-(3-pyridyl)-1-butanone(NNK) and aflatoxin B1(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 CYP2A6Ile300 → Phe, CYP2A6Gly301Ala, CYP2A6Ser369 → Gly, CYP2A13Phe300 → Ile, CYP2A13Ala301 → Gly and CYP2A13Gly369 → Ser 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 CYP2A6Ile300 → Phe and CYP2A6Gly301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe300→Ile and CYP2A13Ala301→Gly replacement introduced remarkable catalytic efficiency decreases. CteIunfYtf iaioPcdni2de Aiontif1co #3yn3G ,v 6ali9ylal 3 r6tae9h s→seiisdge uSn ecei afrsit.iac gClanyonitnfti cicKc leaumfns fdtiilcoeyinc ea:rn#fecf3aey0sc 0eate ,l vtd#ee 3rbra0sot1uitoh san KnVsd mmw a#axe 3nree6dn 9 chV aarmuenassxcei evddmau bleueysen iVstn,. m whaCxu hYvmialPaer2in aAt htCi6eoYS noePspr 2r3pa6A9oth→6sei/t1reG 3t h lepyafl fnaien yKcc tirmsme cawphsoeea rrnttegha eenss etc. e raSontual ewlbyssi tttiiinhc- 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. Ser369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu370 which is crucial for many hydroxylations.
Objective: We have continued previous work in which we said that # 117 and # 372 amino acids contributive to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1- (3-pyridyl) -1-butanone (NNK) and aflatoxin B1 (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 CYP2A6Ile300 → Phe, CYP2A6Gly301Ala, CYP2A6Ser369 → Gly, CYP2A13Phe300 → Ile, CYP2A13Ala301 → Gly and CYP2A13Gly369 → Ser were generated by site-directed mutagenesis / baculovirus-Sf9 insect cells expression. of nicotine 5’hydroxylatin by wild type and mutant CYP2A proteins was performed. Results: All amino acid residue substitutions at 300, 301 and 369 While CYP2A6Ile300 → Phe and CYP2A6Gly301 → Ala mutations had not catalytic catalytic efficiency increases compared to that for wild type CYP2A6, CYP2A13Phe300 → Ile and CYP2A13Ala301 → Gly replacement introduced significant reductions in catalytic activity. CteIunfYtf iaioPcdni2de Aiontif1co # 3yn3G, v 6ali9ylal 3 r6tae9h s → seiisdge uSn ecei afrsit.iac gClanyonitnfti cicKc leaumfns fdtiilcoeyinc ea: rn # fecf3aey0sc 0eate, l vtd # ee 3rbra0sot1uitoh san KnVsd mmw a # axe 3nree6dn 9 chV aarmuenassxcei evddmau bleueysen iVstn ,. m whaCxu hYvmialPaer2in aAt htCi6eoYS noePspr 2r3pa6A9oth → 6sei / t1reG 3t h lepyafl fnaien yKcc tirmsme cawphsoeea rrnttegha eenss etc. e raSontual ewlbyssi tttiiinhc-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. Ser369 to Gly substitution was affected nicotine bindin g by creating more space and conformational flexibility for the nearby residues, such as Leu370 which is crucial for many hydroxylations.