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. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substi-tution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser369→Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly369→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. 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.