A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases

The O⁶-methylguanine-DNA methyltransferase (MGMT) repairs mutagenicand carcinogenic O⁶-alkylguanine in DNA by accepting stoichiometricallythe alkyl group from the base. Although the mouse MGMT is largerthan the human protein because of an additional tetrapeptidesequence, these proteins are 70% homologous. Recombinant MGMTsof the human, the mouse and a mouse mutant with the tetrapeptidedeleted were purified to homogeneity from Escherichia coli.The N-terminal amino acid sequences of these proteins are identicalto those predicted from the nucleotide sequences, and theirmolecular masses deter mined by SDS-PAGE agreed with the predictedvalues. However, the observed isoelectric points of 9.3, 9.2and 9.3, for the human, mouse and mutant mouse proteins respectivelywere significantly different from the values, 8.09, 7.47 and7.49 calculated from the amino acid composition. The extinctioncoefficients E_1%^{280 nm} of human, mouse and mutant mouse proteinwere calculated from amino acid composition to be 18.2, 11.1and 11.3 respectively. These values agree fairly well with calculatedvalues. Human and wild-type mouse MGMTs react with the alkylatedbase in a synthetic DNA substrate poly(dC, dG, m⁶dG) with comparablesecond-order rate constants of 2.2x10⁸ and 3.7x10⁸ 1/M/min at37°C respectively and were inactivated by O⁶-benzylguanineat similar rates. The initial reaction rate (K_in) and rate ofinactivation (k_inact) constants for reaction with the base werecalculated to be 1.8x10^–4 M and 1.4x10^–3/s for thehuman protein, 2.3x10^–4 M and 1.1x10^–3/s for thewild-type mouse protein, and 2.1x10^–4 and 1.4x10^–3/sfor the mutant mouse protein respectively. The MGMTs were inactivatedto the extent of 55—65% after heating at 50°C in 20mMTris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However,in the presence of DNA (200 µg/ml), only 25—35%of the protein was inactivated. Both DNA and RNA inhibited allthree enzymes in a concentration-dependent fashion, althoughDNA was a better inhibitor than RNA. High salt (0.2 M NaCl)inhibited human MGMT by 80%, while the wild-type and the mutantmouse MGMTs were inhibited by 55%. The human protein had higheraffinity for binding to duplex DNAs than the mouse proteins.