Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA(1).gpNu1(2) holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit Tomka and Catalano (1993) Biochemistry 32, 11992-11997; Hwang et al. (1996) Biochemistry 35, 2796-2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of lambda terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTP hydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (k(cat) approximately 37 min(-)(1); K(m) approximately 500 microM). Moreover, while GTP stimulates ATP hydrolysis (apparent K(D) approximately 135 microM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.