PMID- 19146391
OWN - NLM
STAT- MEDLINE
DCOM- 20090303
LR  - 20131121
IS  - 1520-4995 (Electronic)
IS  - 0006-2960 (Linking)
VI  - 48
IP  - 5
DP  - 2009 Feb 10
TI  - Biochemical characterization of recombinant hepatitis C virus nonstructural 
      protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity.
PG  - 906-16
LID - 10.1021/bi801747p [doi]
AB  - While nonstructural protein 4B (NS4B) from hepatitis C virus (HCV) is absolutely 
      required for viral propagation, a full understanding of the enzymatic properties 
      of this protein is lacking. Previous studies suggest that NS4B is located at the 
      endoplasmic reticulum and that the protein structure consists of four central 
      transmembrane domains with the N- and C-termini located in the cytoplasm of the 
      host cell. To characterize the enzymatic activity of NS4B, the full-length 
      protein with a C-terminal His tag was expressed in Sf9 insect cells and 
      stabilized with nonionic detergents during purification. Chemical cross-linking 
      experiments using GTP-gamma-azidoanilide and ATP-gamma-azidoanilide and 
      equilibrium binding analyses with GTPgammaS and ATPgammaS show that both GTP and 
      ATP are bound by NS4B, with ATP displaying a higher affinity. Analyses of 
      enzymatic reactions catalyzed by NS4B indicate that the terminal phosphate groups 
      of ATP, GTP, and GDP are removed to produce ADP, GDP, and GMP, respectively. The 
      k(cat) for hydrolysis of GTP by purified NS4B compared favorably with the k(cat) 
      for hydrolysis of GTP by Ras-p21 in the absence of GTPase activating proteins 
      (GAPs). In addition to the hydrolysis of NTP and NDP substrates, adenylate kinase 
      activity was detected in purified preparations of NS4B with the reverse reaction 
      2ADP --> ATP + ADP, yielding a larger k(cat) compared to that of the forward 
      reaction ATP + AMP --> 2ADP. These studies suggest that HCV NS4B possesses both 
      adenylate kinase activity and nucleotide hydrolase activity. Mutation of amino 
      acids in the Walker A and B motifs of NS4B resulted in decreased affinity for 
      both GTPgammaS and ATPgammaS as well as decreased ATP hydrolysis and AK activity.
FAU - Thompson, Aaron A
AU  - Thompson AA
AD  - Department of Biochemical Pharmacology, La Jolla Laboratories, Pfizer Global 
      Research and Development Inc., San Diego, California 92121, USA.
FAU - Zou, Aihua
AU  - Zou A
FAU - Yan, Jiangli
AU  - Yan J
FAU - Duggal, Rohit
AU  - Duggal R
FAU - Hao, Weidong
AU  - Hao W
FAU - Molina, David
AU  - Molina D
FAU - Cronin, Ciaran N
AU  - Cronin CN
FAU - Wells, Peter A
AU  - Wells PA
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (NS4 protein, hepatitis C virus)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Viral Nonstructural Proteins)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - 8L70Q75FXE (Adenosine Triphosphate)
RN  - EC 2.7.4.3 (Adenylate Kinase)
RN  - EC 3.- (Hydrolases)
SB  - IM
MH  - Adenosine Triphosphate/chemistry/*metabolism
MH  - Adenylate Kinase/chemistry/*metabolism
MH  - Amino Acid Sequence
MH  - Enzyme Activation/physiology
MH  - Guanosine Triphosphate/chemistry/*metabolism
MH  - Hepacivirus/*enzymology
MH  - Hydrolases/chemistry/metabolism
MH  - Hydrolysis
MH  - Molecular Sequence Data
MH  - Recombinant Proteins/chemistry/*metabolism
MH  - Viral Nonstructural Proteins/chemistry/*metabolism
EDAT- 2009/01/17 09:00
MHDA- 2009/03/04 09:00
CRDT- 2009/01/17 09:00
PHST- 2009/01/17 09:00 [entrez]
PHST- 2009/01/17 09:00 [pubmed]
PHST- 2009/03/04 09:00 [medline]
AID - 10.1021/bi801747p [pii]
AID - 10.1021/bi801747p [doi]
PST - ppublish
SO  - Biochemistry. 2009 Feb 10;48(5):906-16. doi: 10.1021/bi801747p.