The multifunctional 39 kDa Escherichia coli Ada protein (O6-methylguanine-DNA 
methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric 
globular polypeptide with two distinct alkylacceptor activities located in two 
domains. The two domains are of nearly equal size and are connected by a hinge 
region. The Ada protein accepts stoichiometrically the alkyl group from 
O6-alkylguanine in DNA at the Cys-321 residue and from alkyl phosphotriester at 
the Cys-69 residue. This protein functions in DNA repair by direct dealkylation 
of mutagenic O6-alkylguanine. The protein methylated at Cys-69 becomes a 
transcriptional activator of the genes in the ada regulon, including its own. 
Each of the two domains functions independently as an alkyl acceptor. The 
purified homogeneous protein is unstable at 37 degrees C and spontaneously loses 
about 30% of its secondary structure in less than 30 min concomitant with a 
complete loss of activity. However, sedimentation equilibrium studies indicated 
that the inactive protein remains in the monomeric form without aggregation. 
Furthermore, electrospray mass spectroscopic analysis indicated the absence of 
oxidation of the inactive protein. This temperature-dependent inactivation of 
the Ada protein is inhibited by DNA. In the presence of increasing 
concentrations of urea or guanidine, the protein gradually loses more than 80% 
of its structure. The two alkyl acceptor activities appear to be differentially 
sensitive to unfolding and the phosphotriester methyltransferase activity is 
resistant to 7 M urea. The partial or complete unfolding induced by urea or 
guanidine is completely reversed within seconds by removal of the denaturant. 
The heat-coagulated protein can also be restored to full activity by cycling it 
through treatment with 8 M urea or 6 M guanidine. These results suggest that the 
nascent or unfolded Ada polypeptide folds to a metastable form which is active 
and that the thermodynamically stable structure is partially unfolded and 
inactive.