BRENDA - Enzyme Database show
show all sequences of 6.3.5.7

Mechanistic studies of reaction coupling in Glu-tRNAGln amidotransferase

Horiuchi, K.Y.; Harpel, M.R.; Shen, L.; Luo, Y.; Rogers, K.C.; Copeland, R.A.; Biochemistry 40, 6450-6457 (2001)

Data extracted from this reference:

Activating Compound
Activating Compound
Commentary
Organism
Structure
additional information
activation of glutaminase activity by ATP or ATP-gammaS together with Glu-tRNAGln, results either from an allosteric effect due simply to binding of these analogues to the enzyme or from some structural changes that attend ATP or ATP-gammaS hydrolysis
Streptococcus pyogenes
Inhibitors
Inhibitors
Commentary
Organism
Structure
2'-O-(trinitrophenyl)adenosine 5'-triphosphate
IC50: 2.4 mM
Streptococcus pyogenes
3'-O-(trinitrophenyl)adenosine 5'-triphosphate
IC50: 2.4 mM
Streptococcus pyogenes
adenosine 5'-[beta,gamma-methylene]triphosphate
IC50: 2.3 mM
Streptococcus pyogenes
ADP
IC50: 0.026 mM
Streptococcus pyogenes
ATP-gammaS
IC50: 0.19 mM
Streptococcus pyogenes
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0002
-
L-glutamyl-tRNAGlu
-
Streptococcus pyogenes
0.0159
-
Gln
-
Streptococcus pyogenes
0.117
-
ATP
-
Streptococcus pyogenes
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ATP + Glu-tRNAGln + L-glutamine
Streptococcus pyogenes
organisms lacking Gln-tRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGln through the transamidation activity of Glu-tRNAGln amidotransferase. The enzyme hydrolyzes Gln to Glu and NH3, using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis
ADP + phosphate + Gln-tRNAGln + L-glutamate
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Streptococcus pyogenes
-
recombinant enzyme expressed in Escherichia coli
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + Glu-tRNAGln + L-glutamine
organisms lacking Gln-tRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGln through the transamidation activity of Glu-tRNAGln amidotransferase. The enzyme hydrolyzes Gln to Glu and NH3, using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis
492302
Streptococcus pyogenes
ADP + phosphate + Gln-tRNAGln + L-glutamate
-
-
-
?
ATP + Glu-tRNAGln + L-glutamine
organisms lacking Gln-tRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGln through the transamidation activity of Glu-tRNAGln amidotransferase. The enzyme hydrolyzes Gln to Glu and NH3, using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis
492302
Streptococcus pyogenes
ADP + phosphate + Gln-tRNAGln + L-glutamate
-
492302
Streptococcus pyogenes
?
ATP-gammaS + Glu-tRNAGln + L-glutamine
-
492302
Streptococcus pyogenes
? + phosphate + Gln-tRNAGln + L-glutamate
-
492302
Streptococcus pyogenes
?
additional information
in absence of the amido acceptor, Glu-tRNAGln, the enzyme has basal glutaminase activity that is unaffected by ATP
492302
Streptococcus pyogenes
?
-
-
-
-
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.51
-
Gln
-
Streptococcus pyogenes
0.59
-
ATP
-
Streptococcus pyogenes
IC50 Value
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.026
-
IC50: 0.026 mM
Streptococcus pyogenes
ADP
0.19
-
IC50: 0.19 mM
Streptococcus pyogenes
ATP-gammaS
2.3
-
IC50: 2.3 mM
Streptococcus pyogenes
adenosine 5'-[beta,gamma-methylene]triphosphate
2.4
-
IC50: 2.4 mM
Streptococcus pyogenes
2'-O-(trinitrophenyl)adenosine 5'-triphosphate
2.4
-
IC50: 2.4 mM
Streptococcus pyogenes
3'-O-(trinitrophenyl)adenosine 5'-triphosphate
Activating Compound (protein specific)
Activating Compound
Commentary
Organism
Structure
additional information
activation of glutaminase activity by ATP or ATP-gammaS together with Glu-tRNAGln, results either from an allosteric effect due simply to binding of these analogues to the enzyme or from some structural changes that attend ATP or ATP-gammaS hydrolysis
Streptococcus pyogenes
IC50 Value (protein specific)
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.026
-
IC50: 0.026 mM
Streptococcus pyogenes
ADP
0.19
-
IC50: 0.19 mM
Streptococcus pyogenes
ATP-gammaS
2.3
-
IC50: 2.3 mM
Streptococcus pyogenes
adenosine 5'-[beta,gamma-methylene]triphosphate
2.4
-
IC50: 2.4 mM
Streptococcus pyogenes
2'-O-(trinitrophenyl)adenosine 5'-triphosphate
2.4
-
IC50: 2.4 mM
Streptococcus pyogenes
3'-O-(trinitrophenyl)adenosine 5'-triphosphate
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
2'-O-(trinitrophenyl)adenosine 5'-triphosphate
IC50: 2.4 mM
Streptococcus pyogenes
3'-O-(trinitrophenyl)adenosine 5'-triphosphate
IC50: 2.4 mM
Streptococcus pyogenes
adenosine 5'-[beta,gamma-methylene]triphosphate
IC50: 2.3 mM
Streptococcus pyogenes
ADP
IC50: 0.026 mM
Streptococcus pyogenes
ATP-gammaS
IC50: 0.19 mM
Streptococcus pyogenes
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0002
-
L-glutamyl-tRNAGlu
-
Streptococcus pyogenes
0.0159
-
Gln
-
Streptococcus pyogenes
0.117
-
ATP
-
Streptococcus pyogenes
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ATP + Glu-tRNAGln + L-glutamine
Streptococcus pyogenes
organisms lacking Gln-tRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGln through the transamidation activity of Glu-tRNAGln amidotransferase. The enzyme hydrolyzes Gln to Glu and NH3, using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis
ADP + phosphate + Gln-tRNAGln + L-glutamate
-
-
?
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + Glu-tRNAGln + L-glutamine
organisms lacking Gln-tRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGln through the transamidation activity of Glu-tRNAGln amidotransferase. The enzyme hydrolyzes Gln to Glu and NH3, using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis
492302
Streptococcus pyogenes
ADP + phosphate + Gln-tRNAGln + L-glutamate
-
-
-
?
ATP + Glu-tRNAGln + L-glutamine
organisms lacking Gln-tRNA synthetase produce Gln-tRNAGln from misacylated Glu-tRNAGln through the transamidation activity of Glu-tRNAGln amidotransferase. The enzyme hydrolyzes Gln to Glu and NH3, using the latter product to transamidate Glu-tRNAGln in concert with ATP hydrolysis
492302
Streptococcus pyogenes
ADP + phosphate + Gln-tRNAGln + L-glutamate
-
492302
Streptococcus pyogenes
?
ATP-gammaS + Glu-tRNAGln + L-glutamine
-
492302
Streptococcus pyogenes
? + phosphate + Gln-tRNAGln + L-glutamate
-
492302
Streptococcus pyogenes
?
additional information
in absence of the amido acceptor, Glu-tRNAGln, the enzyme has basal glutaminase activity that is unaffected by ATP
492302
Streptococcus pyogenes
?
-
-
-
-
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.51
-
Gln
-
Streptococcus pyogenes
0.59
-
ATP
-
Streptococcus pyogenes
Other publictions for EC 6.3.5.7
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
745335
Mailu
Plasmodium apicoplast Gln-tRN ...
Plasmodium berghei, Plasmodium berghei ANKA, Plasmodium falciparum
J. Biol. Chem.
290
29629-29641
2015
-
-
-
-
-
-
-
-
2
-
-
-
-
3
-
-
-
-
-
-
-
-
6
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
6
1
-
-
-
-
-
-
-
-
-
1
1
-
-
-
745612
Dewage
Computational analysis of amm ...
Staphylococcus aureus, Staphylococcus aureus ATCC 700699
J. Phys. Chem. B
119
3669-3677
2015
-
-
-
1
4
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
743955
Chongdar
Preliminary X-ray crystallogr ...
Escherichia coli
Acta Crystallogr. Sect. F
70
922-927
2014
-
-
-
1
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
744268
Echevarria
Glutamyl-tRNAGln amidotransfe ...
Mus musculus
Biochem. J.
460
91-101
2014
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
745552
Hadd
Coevolution of specificity de ...
Saccharomyces cerevisiae
J. Mol. Biol.
426
3619-3633
2014
-
-
-
-
1
-
-
4
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
2
-
-
-
-
-
1
1
-
2
2
728154
Grant
The structure of yeast glutami ...
Thermotoga maritima
J. Mol. Biol.
425
2480-2493
2013
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
728391
Huot
Gln-tRNAGln synthesis in a dyn ...
Helicobacter pylori
Nucleic Acids Res.
39
9306-9315
2011
1
-
-
-
-
-
-
3
-
-
-
1
-
1
-
-
1
-
-
-
-
-
1
-
-
-
-
2
-
-
-
1
-
-
-
1
-
-
1
-
-
-
-
-
-
3
-
-
-
1
-
-
-
1
-
-
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
728668
O'Donoghue
Rational design of an evolutio ...
Methanothermobacter thermautotrophicus
Proc. Natl. Acad. Sci. USA
108
20485-20490
2011
-
-
-
-
1
-
-
4
-
-
-
-
-
1
-
-
1
-
-
-
-
-
3
-
1
-
-
4
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
4
-
-
-
-
-
-
-
1
-
-
-
-
3
-
1
-
-
4
1
-
-
-
-
-
-
-
4
4
709508
Chatani
A simple turbidimetric method ...
Staphylococcus aureus
J. Microbiol. Methods
80
117-122
2010
-
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
714451
Balg
Inhibition of Helicobacter pyl ...
Helicobacter pylori
Bioorg. Med. Chem.
18
7868-7872
2010
-
-
-
-
-
-
7
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
-
-
-
7
-
-
-
-
-
-
-
-
-
-
7
7
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
716331
Ito
Two enzymes bound to one trans ...
Thermotoga maritima
Nature
467
612-616
2010
-
-
1
1
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
716371
Rampias
The archaeal transamidosome fo ...
Methanothermobacter thermautotrophicus
Nucleic Acids Res.
38
5774-5783
2010
-
-
1
-
-
-
-
3
-
-
-
-
-
5
-
-
1
-
-
-
-
-
2
1
1
-
-
2
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
1
-
-
-
-
2
1
1
-
-
2
-
-
-
-
-
-
-
-
3
3
706514
Nagao
Biogenesis of glutaminyl-mt tR ...
Homo sapiens
Proc. Natl. Acad. Sci. USA
106
16209-16214
2009
-
-
-
-
-
-
-
-
1
-
1
-
-
3
-
-
-
-
-
-
-
-
3
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
3
-
-
-
-
-
-
-
-
-
3
3
-
-
-
-
-
-
-
-
-
1
3
-
-
-
692759
Balg
Inhibition of Helicobacter pyl ...
Helicobacter pylori
J. Am. Chem. Soc.
130
3264-3265
2008
-
-
-
-
-
-
8
-
-
-
-
2
-
2
-
-
-
-
-
-
-
-
2
1
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
8
8
-
-
-
-
2
-
-
-
-
-
-
-
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
673689
Namgoong
Co-evolution of the archaeal t ...
Methanothermobacter thermautotrophicus
FEBS Lett.
581
309-314
2007
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
674787
Sheppard
The Helicobacter pylori amidot ...
Helicobacter pylori
J. Biol. Chem.
282
11866-11873
2007
-
-
-
-
3
-
-
7
-
-
-
-
-
1
-
-
1
-
-
-
-
-
2
-
-
-
-
9
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
7
-
-
-
-
-
-
-
1
-
-
-
-
2
-
-
-
-
9
-
-
-
-
-
-
-
-
-
-
662450
Feng
Gln-tRNAGln formation from Glu ...
Methanothermobacter thermautotrophicus
J. Biol. Chem.
280
8150-8155
2005
-
-
1
-
7
-
-
-
-
-
-
-
-
4
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
7
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
492304
Harpel
Mutagenesis and mechanism-base ...
Streptococcus pyogenes
Biochemistry
41
6398-6407
2002
-
-
-
-
-
-
1
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
492305
Kwak
Expression, purification, and ...
Geobacillus stearothermophilus
Mol. Cells
14
374-381
2002
-
-
1
1
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
492302
Horiuchi
Mechanistic studies of reactio ...
Streptococcus pyogenes
Biochemistry
40
6450-6457
2001
1
-
-
-
-
-
5
3
-
-
-
1
-
1
-
-
-
-
-
-
-
-
4
-
-
-
-
2
-
-
-
-
-
-
5
1
-
-
-
-
-
-
5
5
-
3
-
-
-
1
-
-
-
-
-
-
-
-
4
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
492308
Salazar
A dual-specific Glu-tRNA(Gln) ...
Acidithiobacillus ferrooxidans
FEBS Lett.
500
129-131
2001
-
-
1
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
492303
Curnow
Glu-tRNAGln amidotransferase: ...
Bacillus subtilis
Proc. Natl. Acad. Sci. USA
94
11819-11826
1997
-
-
1
-
-
-
-
-
-
1
3
1
-
5
-
-
1
-
-
-
1
-
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
1
3
1
-
-
-
1
-
-
1
-
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
492291
Vothknecht
-
Charging of both, plastidial t ...
Tetradesmus obliquus
Z. Naturforsch. C
50
789-795
1995
-
-
-
-
-
-
-
-
1
-
-
1
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
492306
Jahn
Purification and functional ch ...
Chlamydomonas reinhardtii
J. Biol. Chem.
265
8059-8064
1990
-
-
-
-
-
-
1
-
-
1
2
-
-
1
-
-
1
-
-
-
-
-
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
1
2
-
-
-
-
1
-
-
-
-
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
492310
Strauch
Characterization of the glutam ...
Bacillus subtilis
J. Bacteriol.
170
916-920
1988
-
-
-
-
-
-
2
2
-
-
-
-
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
2
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
492307
Zalkin
Glu-tRNAGln amidotransferase ...
Bacillus subtilis
Methods Enzymol.
113
303-305
1985
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-