Jq. Hang et al., ATPase center of bacteriophage lambda terminase involved in post-cleavage stages of DNA packaging: Identification of ATP-interactive amino acids, J MOL BIOL, 302(4), 2000, pp. 777-795
Terminase is the enzyme that mediates lambda DNA packaging into the viral p
rohead. The large subunit of terminase, gpA (641 amino acid residues), has
a high-affinity ATPase activity (K-m = 5 mu M). To directly identify gpA's
ATP-interacting amino acids, holoterminase bearing a His(6)-tag at the C te
rminus of gpA was UV-crosslinked with 8-N-3-[alpha-P-32]ATP. Tryptic peptid
es from the photolabeled terminase were purified by affinity chromatography
and reverse-phase HPLC. Two labeled peptides of gpA were identified. Amino
acid sequencing failed to show the tyrosine residue of the first peptide,
E(43)SAY(46)QEGR(50), or the lysine of the second peptide, V(80)GYSK(84)MLL
(87), indicating that Y-46 and K-84 were the 8-N-3-ATP-modified amino acids
. To investigate their roles in lambda DNA packaging, Y-46 was changed to E
, A, and F, and K-84 was changed to E and A. Purified His(6)-tagged termina
ses with changes at residues 46 and 84 lacked the gpA high-affinity ATPase
activity, though the cos cleavage and cohesive end separation activities we
re near to those of the wild-type enzyme. In virion assembly reactions usin
g virion DNA as a packaging substrate, the mutant terminases showed severe
defects. In summary, the results indicate that Y-46 and K-84 are part of th
e high-affinity ATPase center of gpA, and show that this ATPase activity is
involved in the post-cos cleavage stages of lambda DNA packaging. (C) 2000
Academic Press.