Kinetic Characterization of the GTPase Activity of Phage Terminase: Evidence for Communication between the Two "NTPase" Catalytic
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- 作者：Liping Woods and Carlos Enrique Catalano
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：November 2, 1999
- 年：1999
- 卷：38
- 期：44
- 页码：14624 - 14630
- 全文大小：100K
- 年卷期：v.38,no.44(November 2, 1999)
- ISSN：1520-4995
文摘
The terminase enzyme from bacteriophage 
is responsible for the insertion of viral DNA intothe confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3kDa) and gpNu1 (20.4 kDa) isolated as a gpA1·gpNu12 holoenzyme complex. Lambda terminase possessesa site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activitythat must work in concert to effect genome packaging. We have previously characterized the ATPaseactivity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka andCatalano (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 GTPhydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activityof terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and isstrongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTPhydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs atboth subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis ofGTPase activity (kcat 
37 min-1; Km 500 
M). Moreover, while GTP stimulates ATP hydrolysis(apparent KD 135 M for GTP binding), all of the adenosine nucleotides examined strongly inhibit theGTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites interminase holoenzyme communicate, and we propose a model describing allosteric interactions betweenthe two sites. The biological significance of this interaction with respect to the assembly and disassemblyof the multiple nucleoprotein packaging complexes required for virus assembly is discussed.
is responsible for the insertion of viral DNA intothe confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3kDa) and gpNu1 (20.4 kDa) isolated as a gpA1·gpNu12 holoenzyme complex. Lambda terminase possessesa site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activitythat must work in concert to effect genome packaging. We have previously characterized the ATPaseactivity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka andCatalano (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 GTPhydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activityof terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and isstrongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTPhydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs atboth subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis ofGTPase activity (kcat 37 min-1; Km 500 M). Moreover, while GTP stimulates ATP hydrolysis(apparent KD 135 M for GTP binding), all of the adenosine nucleotides examined strongly inhibit theGTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites interminase holoenzyme communicate, and we propose a model describing allosteric interactions betweenthe two sites. The biological significance of this interaction with respect to the assembly and disassemblyof the multiple nucleoprotein packaging complexes required for virus assembly is discussed.