KINETIC AND MUTATIONAL DISSECTION OF THE 2 ATPASE ACTIVITIES OF TERMINASE, THE DNA PACKAGING ENZYME OF BACTERIOPHAGE-LAMBDA

Terminase, the DNA packaging enzyme of bacteriophage lambda, is a hete romultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the lambda Nul and A genes, respectively. Sequence comparisons indicate t hat both gpNul and gpA have a match to the P-loop motif of ATPase cent ers, which is a glycine-rich segment followed by a lysine, By site-spe cific mutagenesis, we changed the lysines of the putative P-loops of g pNul (K-35) and gpA (K-497) to arginine, alanine, or aspartic acid, an d studied the mutant enzymes by kinetic analysis and photochemical cro ss-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-t ype terminase were covalently modified with 8-N-3-[P-32]ATP in the pre sence of UV light. Saturation occurred with apparent dissociation cons tants of 508 and 3.5 mu M for gpNu1 and gpA, respectively. ATPase assa ys showed two activities: a low-affinity activity (K-m = 469 mu M), an d a high-affinity activity (K-m = 4.6 mu M). The gpNu1 K(35)A and gpNu l K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recover ed when 10 times more DNA was added, suggesting that the primary defec t of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase, ATPase assays and photolabeling of the gpA K(497)A and gpA K497D mutant terminases showed reduced affinity f or ATP at the high-affinity site which was not restored by increased D NA, In summary, the results :indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA .