Minimum size of active human O⁶-methylguanine-dna methyltransference (MGMT); substrate binding parallels mgmt activity

O⁶-methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, repairs the mutagenic adduct O⁶-alkylguanine by accepting the alkyl group on a unique cysteine residue in DNA. In an effort to identify the minimum size required for MGMT activity, its cDNA was sequentially deleted at one or both termini and the methyltransferase activities of the encoded polypeptides were determined. Up to 8 and 31 residues could be removed from the NH₂- and COO^--termini of the protein, respectively, without loss of activity. Although, removal of residue 9 (Arg) from the NH₂-terminus or residue 32 (Leu) from the COO^--terminus abolished activity, these residues are not essential for activity in the full length protein, but may by involved in maintaining structure. The second order rate constant of the MGMT reaction (5×10⁷ M^- min^-1) was not significantly altered in the active truncated mutants. Specific complexes between MGMT's and O⁶-methylguanine-containing oligonucleotide were only observed in electrophoretic mobility shift assays with active, truncated proteins. Furthermore, conservative substitution of the alkylacceptor cysteine with serine induced a significant conformational change associated with loss of both methyltransferase activity and substrate binding.