Purification and some properties of human DNA- O6-methylguanine methyltransferase.

DNA-O6-methylguanine methyltransferase was purified from the nuclear fraction of fresh human placenta using ammonium sulphate precipitation, gel filtration, affinity chromatography on DNA-cellulose and hydroxyapatite. The methyltransferase preparation was approximately 1-2% pure based on specific activity, and was free of nucleic acids. The protein reacts stoichiometrically with O6-methylguanine in DNA with apparent second-order kinetics. The human methyltransferase has a pH optimum of about 8·5, similar to that of the corresponding rat and mouse proteins. NaCl inhibits the reaction in a concentration-dependent fashion. The human protein, like the rodent and E. coli methyltransferases, needs no cofactor. While lmM MnCl2, lmM spermidine, 5mM MgCl2 and 10 mM EDTA individually do not significantly inhibit the initial rate of reaction, the protein is nearly completely inactive in 5 mM A1C13 or FeCl2 or 10 mM spermidine. The initial rate of reaction increases as a function of temperature at least up to 42°. The reaction is inhibited by DNA in a concentration-dependent manner, with singlestranded DNA being more inhibitory than duplex DNA.

Site-directed mutagenesis to determine essential residues of ribulosebisphosphate carboxylase of Rhodospirillum rubrum.

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ~40% and ~18% as active as the native carboxylase, essentially ruling out His-291 in the Rhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.