COMPARATIVE-STUDIES OF THE PHAGE T2 AND T4 DNA (N-6-ADENINE)METHYLTRANSFERASES - AMINO-ACID CHANGES THAT AFFECT CATALYTIC ACTIVITY

The bacteriophage T2 and T4 dam genes code for a DNA (N-6-adenine)meth yltransferase (MTase), Nonglucosylated, hydroxymethylcytosine-containi ng T2gt(-) virion DNA has a higher level of methylation than T4gt(-) v irion DNA does, To investigate the basis for this difference, we compa red the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed t hat the same amounts of MTase protein were produced after infection wi th T2 and T4. Kinetic analyses with purified homogeneous enzymes showe d that the two MTases had similar K-m values for the methyl donor, S-a denosyl-L-methionine, and for substrate DNA, In contrast, they had dif ferent k(cat) values (twofold higher for T2 Dam MTase), We suggest tha t this difference can account for the ability of T2 Dam to methylate v iral DNA in vivo to a higher level than does T4 Dam, Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T 4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is resp onsible for increased catalytic activity, The results of these analyse s showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and n oncanonical sites, Moreover, a single substitution of either residue 2 0 or 26 was sufficient to increase the k(cat) of T4 Dam.