Substrate Specificity and Chemical Mechanism for the Reaction Catalyzed by Glutamine Kinase

Article abstract of DOI:10.1021/acs.biochem.8b00811

Campylobacter jejuni, a leading cause of gastroenteritis worldwide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (l-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of l-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of l-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated (Enz-X-P_β-P_γ) and phosphorylated (Enz-X-P_β) intermediates. In the absence of l-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reaction within β-[¹⁸O₄]-ATP in support of the formation of the Enz-X-P_β-P_γintermediate. In the absence of ATP, the enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between l-glutamine phosphate and [¹⁵N-amide]-l-glutamine in direct support of the Enz-X-P_βintermediate. The active site nucleophile has been identified as His-737 based on the lack of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was shown to also catalyze the phosphorylation of d-glutamine, γ-l-glutamyl hydroxamate, γ-l-glutamyl hydrazide, and β-l-aspartyl hydroxamate, in addition to l-glutamine.

Full text of DOI:10.1021/acs.biochem.8b00811

Subscriber access provided by Kaohsiung Medical University
Article
Substrate Specificity and Chemical Mechanism
for the Reaction Catalyzed by Glutamine Kinase
Zane W Taylor, Alexandra R Chamberlain, and Frank M. Raushel
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00811 • Publication Date (Web): 24 Aug 2018
Downloaded from http://pubs.acs.org on August 25, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 30
Biochemistry
1
2
3
4
5
6
7
8
9
Substrate Specificity and Chemical Mechanism for the
Reaction Catalyzed by Glutamine Kinase
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Zane W. TaylorΨ, Alexandra R. Chamberlainω,
and
Frank M. RaushelΨ,ω,*
ΨDepartment of Biochemistry & Biophysics, Texas A&M University, College Station,
Texas. 77843, USA
ωDepartment of Chemistry, Texas A&M University, College Station, Texas, 77843, USA
1
ACS Paragon Plus Environment

Biochemistry
Page 2 of 30
1
2
3
4
ABSTRACT
5
6
7
8
Campylobacter jejuni, a leading cause of gastroenteritis world wide, has a unique O-
methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide.
Investigations into the biological role of MeOPN have revealed that it contributes to the
pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni.
Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are
required for the biosynthesis of the phosphoramidate modification have been elucidated.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cj1418 (
with the ATP-dependent phosphorylation of the amide nitrogen of
show that Cj1418 catalyzes the phosphorylation of -glutamine through a three-step
reaction mechanism via the formation of covalent pyrophosphorylated (Enz-X-Pβ-Pγ) and
phosphorylated (Enz-X-Pβ) intermediates. In the absence of -glutamine, the enzyme was
L
-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond
L
-glutamine. Here we
L
L
shown to catalyze a positional isotope exchange (PIX) reaction within β-[18O4]-ATP in
support of the formation of the Enz-X-Pβ-Pγ intermediate. In the absence of ATP, the
enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between
L
-
glutamine phosphate and [15N-amide]-
L-glutamine in direct support of the Enz-X-Pβ
intermediate. The active site nucleophile has been identified as His-737 based on the lack
of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was
shown to also catalyze the phosphorylation of D-glutamine, γ-L-glutamyl hydroxamate, γ-L-
glutamyl hydrazide and β-L-aspartyl hydroxamate, in addition to L-glutamine.
2
ACS Paragon Plus Environment

Page 3 of 30
Biochemistry
1
2
3
4
INTRODUCTION
5
6
7
8
9
Campylobacter jejuni, a Gram-negative pathogenic bacterium, is a leading cause of
gastroenteritis in the US.1 While C. jejuni is pathogenic towards humans, this organism is
commensal towards chickens, and as a result contaminated poultry is a common source of
human infection. Like many other Gram-negative bacteria, C. jejuni produces a capsular
polysaccharide (CPS), which is composed of unusually modified sugars that envelops the
organism. The capsular polysaccharides help protect the organism from the environment
and are important for the invasion and colonization of the host organisms.2 Numerous
strains of C. jejuni have been identified and it is currently believed that each strain
produces a unique capsular polysaccharide.3, 4 An O-methyl phosphoramidate (MeOPN)
modification was discovered in the CPS of the NCTC11168 strain of C. jejuni.4, 5 This
modification to the CPS is most unusual since phosphorus-nitrogen bonds are quite rare in
nature.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The gene cluster for the biosynthesis of the CPS for the NCTC11168 strain of C. jejuni
capsule has been identified, and eight of the 35 genes in this cluster appear to be linked
with the presence of the MeOPN modification in the capsular polysaccharide.5, 6 Of the
eight enzymes that appear to be required for the biosynthesis of the MeOPN modification,
two are predicted to be phosphoramidate transferases (Cj1421 and Cj1422) and two are
annotated as SAM-dependent methyltransferases (Cj1419 and Cj1420). The other four
enzymes (Cj1415, Cj1416, Cj1417, and Cj1418) are directly responsible for the formation of
the phosphoramidate moiety. Previously, we have characterized the four enzymes
responsible for the formation of the phosphoramidate.7-9 The first enzyme, Cj1418, is a
novel glutamine kinase that catalyzes the ATP-dependent phosphorylation of L-glutamine
3
ACS Paragon Plus Environment

Biochemistry
Page 4 of 30
1
2
3
4
(1) to form
L
-glutamine phosphate (2).8
L-glutamine phosphate is subsequently used by
5
6
Cj1416, a CTP:phosphoglutamine cytidylyltransferase, to displace pyrophosphate from CTP
7
8
9
to generate CDP-
hydrolyzes CDP-
L
-glutamine (3).7 Cj1417, a γ-glutamyl-CDP-amidate hydrolase,
-glutamine to form L
-glutamate and cytidine diphosphoramidate (4).7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
L
Cj1415, a cytidine diphosphoramidate kinase, catalyzes the phosphorylation of the 3’
hydroxyl group of cytidine diphosphoramidate.9 The phosphoramidate moiety of the 3’-
phospho 5’-cytidine diphosphoramidate cofactor (5) is likely transferred to the capsule and
methylated or, alternatively, methylated and then transferred to the capsule. The reactions
are summarized in Scheme 1.
Scheme 1: Biosynthesis of 3’-phospho-5’-cytidine diphosphoramidate (5) in C. jejuni
NCTC11168.
Of these four enzymes, glutamine kinase (Cj1418), is perhaps the most interesting
mechanistically, as it is responsible for synthesis of the phosphoramidate bond. Very few
enzymes have been shown previously to catalyze the formation of P-N bonds, however two
strategies exist. Creatine kinase and arginine kinase catalyze the ATP dependent
phosphorylation of their respective substrates and generate ADP as a product.10, 11
4
ACS Paragon Plus Environment

Page 5 of 30
Biochemistry
1
2
3
4
5
6
7
8
9
Alternatively, enzymes that are related to tRNA synthetases can also be used to generate
phosphoramidate bonds through a mechanism similar to adenylylation.12 Cj1418 belongs
to a family of kinases that employ an unusual reaction mechanism. Phospho(enol)pyruvate
synthase catalyzes the ATP-dependent phosphorylation of pyruvate, generating PEP, AMP
and phosphate.13 Pyruvate phosphate dikinase catalyzes the ATP-dependent
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
phosphorylation of phosphate and pyruvate to generate AMP, pyrophosphate and PEP.14
Finally, rifampin phosphotransferase catalyzes the phosphorylation of rifampin with
formation of Pi and AMP.15, 16 All three enzymes share a similar protein architecture, and
contain an ATP-grasp domain, a phospho-histidine domain, and a specialized substrate-
binding domain.16-18 These three domains can be identified in Cj1418, where residues 1-
219 comprise the predicted ATP-grasp domain, residues 220-693 is likely the specialized
substrate-binding domain for
phospho-histidine domain.17
L-glutamine, and residues 694-767 form the predicted
This enzyme family catalyzes the phosphorylation of substrates via a mechanism
that requires three distinct chemical steps. In the first step, the enzyme utilizes a histidine
residue to attack ATP at the β-phosphoryl group to liberate AMP and a pyrophosphorylated
enzyme intermediate. In the next step the pyrophosphorylated enzyme is hydrolyzed to
form inorganic phosphate and a phosphorylated enzyme intermediate. In the final step the
phosphorylated enzyme intermediate transfers the phosphate to the acceptor substrate.
These transformations are summarized in Scheme 2 for the reaction catalyzed by Cj1418.
In this investigation we have addressed the reaction mechanism and substrate
profile for the reaction catalyzed by glutamine kinase. The formation of the
pyrophosphorylated and phosphorylated covalent reaction intermediates have been
5
ACS Paragon Plus Environment

Biochemistry
Page 6 of 30
1
2
3
4
5
6
7
8
9
interrogated by utilization of positional isotope exchange (PIX) and molecular isotope
exchange (MIX) techniques.19, 20 The active site histidine residue has been identified by
mutagenesis and the enzyme has been shown to catalyze the phosphorylation of the
hydroxamate and hydrazide derivatives of glutamate and aspartate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2: Proposed reaction mechanism for formation of L-glutamine phosphate by
Cj1418.
MATERIALS and METHODS
Materials. All buffers and salts were purchased from Sigma-Aldrich, unless
otherwise specified. -glutamine (1), γ-L-glutamyl hydroxamate (7), β-L-aspartyl
L
hydroxamate (9), AMP-morpholidate (10), and phosphorus pentachloride were purchased
from Sigma Aldrich. γ-L-glutamyl hydrazide (8) was obtained from Santa Cruz
Biotechnology. 18O-labeled water (97 atom %) was obtained from Cambridge Isotope
Laboratories. [15N-amide]-
L-glutamine was obtained from Merck, Sharp and Dohme,
Canada. -Glutamine (98% pure) was purchased from Acros Organics. The restriction
D
enzyme DpnI was obtained from New England Biolabs. Primestar HS Polymerase was
purchased from Takara Industries. The plasmid used for the expression of Cj1418 from C.
6
ACS Paragon Plus Environment

Page 7 of 30
Biochemistry
1
2
3
4
5
6
jejuni NCTC 11168 was obtained from Professor Christine Szymanski of the University of
Georgia.
7
8
9
Purification of Glutamine Kinase. The plasmid used for the expression of Cj1418
(UniProt: Q0P8J6) with an N-terminal poly-histidine purification tag was used to transform
Rosetta (DE3) Escherichia coli cells by electroporation. Five-mL cultures of LB medium
supplemented with 50 µg/mL kanamycin and 25 µg/mL chloramphenicol were inoculated
with a single colony and grown overnight at 30 oC. These cultures were used to inoculate 1
L of LB medium (50 µg/mL kanamycin and 25 µg/mL chloramphenicol) and then incubated
at 30 oC until an OD600 of ~0.6-0.8 was reached. The cells were induced with 1.0 mM
isopropyl β-thiogalactoside (IPTG), grown for 16 h at 16 oC and then harvested by
centrifugation at 6000 rpm at 4 oC. The resulting cell pellet was re-suspended into loading
buffer (50 mM HEPES/KOH, 300 mM KCl, 20 mM imidazole, pH 8.0) and lysed by
sonication. The total cell lysate was passed through a 0.45 µm filter before being loaded
onto a prepacked 5-mL HisTrap HP (GE Healthcare) nickel affinity column. Protein was
eluted with 50 mM HEPES/KOH, pH 8.0, 300 mM KCl, and 400 mM imidazole over a
gradient of 30 column volumes. Excess imidazole was removed by exchanging the buffer
against 50 mM HEPES/KOH, pH 8.0, 100 mM KCl, using a 20 mL (10 kDa molecular weight
cutoff) concentrator (GE Healthcare). The protein was concentrated to a volume of 3 mL
and loaded onto a GE Healthcare HiLoad 16/600 Sephadex 120 mL size exclusion column
and eluted with 10 mM HEPES/KOH (pH 8.0) and 100 mM KCl. Fractions were assayed for
catalytic activity, pooled, concentrated, and stored at -80 °C until needed.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Construction of H737N Mutant of Glutamine Kinase. His-787 from Cj1418 was
mutated to asparagine. The plasmid used for the expression of wild-type Cj1418 with an N-
7
ACS Paragon Plus Environment

Biochemistry
Page 8 of 30
1
2
3
4
5
6
terminal hexa-histidine tag was used as a template for the construction of the plasmid for
the expression of the H737N mutant. The forward (5’-
7
8
9
GGGGGTGCTAATTCAAATATGGCCATTCGTGC-3’) and reverse primers (5’-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
GCACGAATGGCCATATTTGAATTAGCACCCCC-3’) were used to convert the histidine codon
(CAT) at residue 737 to asparagine (AAT). Primestar HS polymerase was used to amplify
the gene. The reaction used a three-step thermal cycle (98 oC for 10 s, 55 oC for 40 s, and 72
oC for 10 min) and continued through thirty cycles. After amplification, DpnI was used to
digest the template DNA for 2 h at 37 oC. Following DpnI digestion, PCR cleanup (Qiagen)
was performed and the plasmid transformed into E. coli BL21 (DE3) cells. Single colonies
were selected and the DNA sequence of the mutant gene was confirmed.
Determination of Kinetic Constants. The kinetic constants for Cj1418 were
determined using a pyruvate kinase/lactate dehydrogenase/myokinase coupled enzyme
assay by following the oxidation of NADH at 340 nm at 25 oC with a SpectraMax340 UV-
visible spectrophotometer. Assays were performed in 100 mM HEPES/KOH, pH 8.0, and
100 mM KCl. The coupling system contained 2.0 mM PEP, 0.3 mM NADH, 14 mM MgCl2, 10
mM ATP, 8 units/mL lactate dehydrogenase, 8 units/mL adenylate kinase and 8 units/mL
pyruvate kinase (PK). The kinetic constants for D-glutamine (2.0 - 20 mM), γ-L-glutamyl
hydroxamate (0.4 - 4.0 mM), γ-L-glutamyl hydrazide (2.0 – 20 mM), β-L-aspartyl
hydroxamate (5.0 - 40 mM) were all assayed using 250 nM Cj1418. The kinetic constants
were determined by fitting the initial rates to Eqn. 1 using GraFit 5, where υ is the initial
velocity of the reaction, Et is the enzyme concentration, kcat is the turnover number, [A] is
the substrate concentration, and Km is the Michaelis constant.
v/Et = kcat (A) / (A + Km)
(1)
8
ACS Paragon Plus Environment

Page 9 of 30
Biochemistry
1
2
3
4
5
6
7
8
9
Synthesis of [18O4]-Phosphate. The synthesis of [18O4]-Pi (11) was carried out as
reported previously.21 The reaction was conducted in a 10-mL round bottom flask that was
placed in an ice bath. The reaction utilized 1.0 g of water containing 97% 18O, and 2.5 g of
PCl5. PCl5 was slowly added to the water and then heated to 100 °C and allowed to stir for
an additional 2 h. After heating, the reaction was cooled to room temperature, the pH
adjusted to 7.0, and aliquots were flash frozen and stored at -80 °C. The 31P NMR spectrum
of the product indicated that 93% of the phosphate contained four 18O-atoms and 7%
contained three 18O-atoms, for an overall 18O-content of 98%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of β-[18O4]-ATP. The procedure for the synthesis of 18O-labeled ATP
where the β-phosphoryl group contains four 18O-atoms was modified from a previously
reported method.22, 23 AMP morpholidate (10, 1.0 g) and the tributylammonium salt of
[18O4]-phosphate were stirred in dry DMSO (20 mL) at room temperature for three days.
The reaction was diluted with water (100 mL) and then loaded onto a column of DEAE
Sephadex. The β-[18O4]-ADP (12) was eluted from the column using a linear gradient of
triethylammonium bicarbonate (300 mL from 0.01 to 1.0 M) and then concentrated by
rotary evaporation. The labeled ADP (20 mM) was incubated with pyruvate kinase (3
units/mL) and 25 mM phospho(enol)pyruvate (PEP) in 50 mM HEPES/KOH, pH 8.0 for 16
h in a volume of 50 mL. The reaction mixture was loaded onto a column of DEAE Sephadex
and eluted with triethylammonium bicarbonate (600 mL from 0.01 to 1 M). The fractions
containing β-[18O4]-ATP (13) were identified by the absorbance at 260 nm and 31P NMR
spectroscopy. Excess triethylamine removed by evaporation and the product stored at -80
°C. The reactions are summarized in Scheme 3.
9
ACS Paragon Plus Environment

Biochemistry
Page 10 of 30
1
2
3
4
5
6
7
8
9
Synthesis of Hydroxylamine O-Phosphate Ester. O-phosphorylated
hydroxylamine (14) was synthesized using pyruvate kinase.24 Pyruvate kinase (4
units/mL) was incubated with 20 mM ATP, 25 mM hydroxylamine, 30 mM zinc acetate and
100 mM ammonium bicarbonate (pH 8.0). The reaction was monitored by 31P NMR
spectroscopy until all of the ATP was converted to ADP. Once complete, the reaction
mixture was diluted with water and loaded onto a DEAE Sephadex anion exchange resin
and eluted with a linear gradient of triethylammonium bicarbonate (0.01 to 1.0 M).
Fractions containing the O-phosphorylated hydroxylamine were identified by 31P NMR
spectroscopy, concentrated, flash frozen and then stored at -20 °C.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3: Chemical synthesis of β-[18O4]-ADP (12) and enzymatic syntheses of β-[18O4]-
ATP (13) and hydroxylamine O-phosphate ester (14).
Positional Isotope Exchange (PIX). A reaction mixture containing 5 mM β-[18O4]-
ATP (13), 7 mM MgCl2, and 10 μM Cj1418 in 100 mM KCl, 100 mM HEPES/KOH, 50 mM
CHES/KOH, pH 8.0, was incubated at 25 °C. At various times, 500 μL of the reaction
mixture were removed and quenched with 15 mM EDTA and the pH adjusted to 9.5 before
31P NMR analysis. Samples that were not analyzed immediately were flash frozen and
10
ACS Paragon Plus Environment

Page 11 of 30
Biochemistry
1
2
3
4
5
6
7
8
9
stored at -20 °C. Aliquots were collected at 0, 1, 4, 8, 12, and 15 h and the reaction
velocities were determined using Eqn. 2, where X is the fraction of change of the original
ATP pool, and F is the fraction of the equilibrium value for positional isotope exchange in
the ATP pool at time t.25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
υex = (X / ln(1-X)) (ATP0/t) (ln(1-F))
(2)
Molecular Isotope Exchange (MIX). The reaction mixture containing 10 mM L-
glutamine phosphate and 10 mM [15N-amide]-
and 20 μM Cj1418 for 12 h in 100 mM HEPES/KOH, 100 mM KCl (pH 8.0) at 25 °C. The
L-glutamine was incubated with 5 mM MgCl2,
reaction products were analyzed by 15N and 31P NMR spectroscopy. [15N-amide]-
L-
glutamine phosphate was made enzymatically as previously described.8
RESULTS
Substrate Specificity of Cj1418. Cj1418 has previously been identified as a
-glutamine.8
glutamine kinase that catalyzes the ATP-dependent phosphorylation of
L
However, the substrate profile for this enzyme has not been adequately determined. In
addition to -glutamine, 15 structural analogs of -glutamine were tested as potential
substrates for Cj1418 (Scheme S1). Of the compounds tested, only -glutamine (1),
L
L
L
D-
glutamine (6), γ-L-glutamyl hydroxamate (7), γ-L-glutamyl hydrazide (8), and β-L-aspartyl
hydroxamate (9) exhibited substrate activity based on the formation of AMP and Pi
(Scheme 4). The kinetic constants for the phosphorylation of these compounds by Cj1418
are summarized in Table 1.
11
ACS Paragon Plus Environment

Biochemistry
Page 12 of 30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4: Alternate substrates for Cj1418.
Characterization of Reaction Products. The reaction products for the
phosphorylation of the alternate substrates 6-9 were analyzed by 31P NMR spectroscopy
(Figures S1 through S4). The product for the reaction with
D-glutamine (6) exhibited two
new resonances (-3.65 and -4.28 ppm) with the same chemical shifts as reported
previously for the reaction of Cj1418 with
L-glutamine and formation of L-glutamine
phosphate (2).8 These two resonances represent the syn- and anti-conformers of
D
-
glutamine phosphate (Figure S1). With the hydroxamate derivative of L-glutamate (7),
two new resonances were observed at 5.91 ppm (major) and 6.33 ppm (minor) ppm after
the addition of ATP and Cj1418 (in addition to those for AMP and Pi). However, this
product was unstable and a new resonance was observed at 7.92 ppm after ~24 h. The
degradation product was shown to be hydroxylamine-O-phosphate (14) by direct
comparison of authentic 14 by 31P NMR (Figure S2). This result is consistent with the
enzymatic phosphorylation of 7 on the hydroxyl group and the initial formation of
compound 15 (Scheme 5). Formation of 14 is proposed to occur via the intramolecular
attack of the α-amino group of 15 with the C5 carbonyl group and generation of
pyroglutamic acid (16) as shown in Scheme 5. The 13C NMR spectrum of the reaction
mixture confirmed the formation of pyroglutaminic acid (Figure S5a).
12
ACS Paragon Plus Environment

Page 13 of 30
Biochemistry
1
2
3
4
With the hydrazide derivative of L
-glutamine (8) two new 31P NMR resonances were
5
6
7
8
observed with chemical shifts of 5.66 ppm (major) and 5.47 ppm (minor) (Figure S3a).
This product was unstable (17) and subsequently converted to phosphate (Figure S3b).
The 13C NMR spectrum is consistent with the formation of pyroglutamate (Figure S5b).
The fate of the hydrazine is unknown. The reaction product with the hydroxamate of
aspartate (9) exhibited two new 31P NMR resonances with chemical shifts of 6.07 ppm
(major) and 6.44 ppm (minor) (Figure S4a). However, unlike the phosphorylated
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
hydroxamate from
L-glutamate (15) the phosphorylated hydroxamate product (18) from L-
aspartate is stable (Figure S4b). Presumably, this is a direct consequence of the fact that
nucleophilic attack by the α-amino group of 16 is hindered by the steric strain imposed by
the formation of a β-lactam.
Table 1: Kinetic constants for Cj1418 at 25 oC, pH 8.0, and 10 mM ATP.
substrate
kcat (s-1)
Km (mM)
kcat/Km (M-1 s-1)
L
-glutamine (1)
2.5 ± 0.3
1.5 ± 0.1
2.1 ± 0.2
0.41 ± 0.05
nda
0.64 ± 0.06
10.5 ± 1.8
1.5 ± 0.4
17.2 ± 3.8
nda
3900 ± 800
140 ± 30
1400 ± 360
24 ± 6
D
-glutamine (6)
-glutamyl hydroxamate (7)
γ- -glutamyl hydrazide (8)
β- -aspartyl hydroxamate (9)
41
γ-
42
43
L
L
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
L
1.2 ± 0.2b
and, not determined.
bA plot of reaction rate versus substrate concentration was linear up to a concentration of
40 mM
13
ACS Paragon Plus Environment

Biochemistry
Page 14 of 30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5: Reaction products for the phosphorylation of 7, 8, and 9 by Cj1418.
Characterization of Reaction Products with L-Glutamine. It was previously
demonstrated that Cj1418 catalyzes the formation of AMP, Pi, and
L-glutamine phosphate
from ATP, water, and
L
-glutamine.8 The proposed mechanism of action (Scheme 2)
assumes that the β-phosphoryl group of ATP is ultimately found as L-glutamine phosphate,
whereas the γ-phosphoryl group is found as Pi. However, the previous characterization of
the reaction products did not differentiate between the β- and γ–phosphoryl groups in the
two reaction products.8 To unambiguously determine the origin of the two products from
ATP, the reaction was first conducted in 100% [16O]-H2O and then in 50% [18O]-H2O. For
the product Pi, there is a single phosphorus resonance when the reaction was conducted in
unlabeled water (Figure 1a) but two phosphorus resonances, separated by ~0.023 ppm
from one another, when the reaction was conducted in a mixture of 16O- and 18O-H2O
(Figure 1b). The up-field resonance is for the phosphate with a single 18O-label and the
14
ACS Paragon Plus Environment

Page 15 of 30
Biochemistry
1
2
3
4
5
6
down-field resonance is for phosphate without an 18O-label.26 The 31P NMR spectrum of the
reaction products clearly indicates that only the Pi product was labeled with 18O.
7
8
9
In a second experiment, the Cj1418-catalyzed reaction was conducted with L-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
glutamine and a 50/50 mixture of β-[18O4]-ATP (13) and unlabeled ATP. In the 31P NMR
spectrum of the reaction products, a single resonance is observed for phosphate, and four
resonances are observed for the L-glutamine phosphate product (Figure 1c). One pair of
resonances is for the unlabeled product (syn- and anti-conformations) and the other for the
product labeled with 18O. The separation in the chemical shifts for the two pairs of
resonances is 0.072 ppm, consistent with the incorporation of three 18O-atoms, as expected
from the labeled ATP used in this experiment.26 These two experiments clearly indicate
that the β-phosphoryl group is ultimately found in L-glutamine phosphate and that the γ-
phosphoryl group is ultimately found as phosphate. This confirms part of the mechanism
proposed in Scheme 2.
15
ACS Paragon Plus Environment

Biochemistry
Page 16 of 30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. 31P NMR spectra of the products in the reaction catalyzed by Cj1418. (A) 31P
NMR spectrum after incubation of Cj1418 (5 µM) with
L
-glutamine (5 mM) and MgATP (5
-glutamine phosphate, 2.43
mM) at pH 8.0. Resonances at -3.66 and -4.31 ppm are from
L
ppm is from phosphate and 3.91 ppm is from AMP. (B) Same reaction conditions as for
spectrum A, but the reaction was conducted in 50% [18O]-H2O. The 31P NMR resonance for
phosphate is shifted up-field by 0.023 ppm due to the incorporation of a single atom of 18O
in the phosphate product. (C) Same reaction conditions as for spectrum A, except that
unlabeled ATP was mixed with 50% of β-[18O4]-ATP (13). The 31P NMR spectrum for
L-
glutamine phosphate exhibits four resonances. There are two resonances each for the syn-
and anti-conformers that are separated by 0.072 ppm due to the incorporation of 3 atoms
of 18O. In the spectra, the 31P NMR resonance for AMP appears at 3.91 ppm. In spectrum C
the 31P NMR resonance for AMP exhibits two resonance separated by 0.024 ppm due to the
presence of a single atom of 18O from the enzymatic cleavage of the bond between the β-P
and α/β-bridging oxygen in the labeled ATP used in this reaction.
16
ACS Paragon Plus Environment

Page 17 of 30
Biochemistry
1
2
3
4
5
6
Importance of His-737 as Enzyme Nucleophile. The closest homologues of
Cj1418 include PEP synthase, pyruvate phosphate dikinase, and rifampin
7
8
9
phosphotransferase.13, 15, 16, 18 Each of these enzymes has been structurally characterized
and shown to be pyrophosphorylated on a conserved histidine residue that originates from
either a central or C-terminal domain of the protein.16, 18 The multi-protein sequence
alignment of Cj1418 (residues 694-767) with the phospho-histidine domains of these
proteins (Figure S6) identifies His-737 as the residue that is likely to be
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
pyrophosphorylated in the reaction mechanism of Cj1418 (Scheme 2). This residue was
mutated to asparagine (H737N) and the mutant assayed for its ability to phosphorylate
glutamine, however, no activity was detected (kcat ≤ .001 s-1). This result is consistent with
His-737 as being the nucleophile that attacks the β-phosphoryl group of ATP with
formation of a pyrophosphorylated enzyme intermediate.
Detection of Pyrophosphorylated Intermediate. In the proposed reaction
mechanism for Cj1418, the first step is initiated by the nucleophilic attack of the enzyme
(likely His-737) on the β-phosphoryl group of ATP to generate a covalent
pyrophosphorylated intermediate and AMP (Scheme 2a). The significance of this first step
was addressed by measurement of the positional isotope exchange (PIX) reaction using β-
[18O4]-ATP (13) in the absence of added -glutamine.19, 20 To observe a PIX reaction with
L
Cj1418, the formation of the Enz-X-PPi intermediate must be reversible and the α-
phosphoryl group of AMP bound in the active site must be free to rotate. The positional
exchange of the 18O label is outlined in Scheme 6. As the reaction proceeds, the 18O that
originally occupies the α/β-bridge position in the labeled ATP (13) will equilibrate with the
16O in the two α-nonbridge positions to form 19 and 20. At equilibrium, 1/3 of the 18O
17
ACS Paragon Plus Environment

Biochemistry
Page 18 of 30
1
2
3
4
5
6
label will remain in the α/β-bridge position and 2/3 will be in the two α-nonbridge
positions.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 6: Positional isotope exchange reaction with Cj1418 and β-[18O4]-ATP.
The PIX reaction was followed by 31P NMR spectroscopy since the incorporation of
18O into ATP results in a perturbation in the chemical shift for phosphorus and the
magnitude of the shift is dependent on whether the 18-oxygen occupies a bridge or non-
bridge position within the polyphosphate moiety of ATP.26 It has been previously shown
that 18O in the α/β-bridge position of ATP will cause a +0.017 ppm up-field chemical shift
perturbation for the α- and β-phosphoryl groups.26 In the α-nonbridge position there will
be a +0.028 ppm up-field shift perturbation for a single 18-oxygen.26 Thus, as the α/β-
bridge oxygen in the β-[18O4]-ATP scrambles, there will be a change in the chemical shift of
the β-phosphate of approximately -0.017 ppm and an increase in the chemical shift for the
α-phosphate by ~0.011 ppm (0.028-0.017 ppm).
The PIX reaction was initiated by incubation of 5 mM β-[18O4]-ATP (13) and 10 μM
Cj1418. Aliquots were removed, the reaction quenched with EDTA, and the products
analyzed by 31P NMR spectroscopy. The 31P NMR spectra of the α- and β-phosphoryl
18
ACS Paragon Plus Environment

Page 19 of 30
Biochemistry
1
2
3
4
5
6
7
8
9
groups of the labeled ATP, before the addition of the Cj1418 and after 8 h, are presented in
Figure 2. For the β-phosphoryl group, the 31P NMR spectrum (Figure 2c) indicates that in
the original β-[18O4]-ATP, 86% is labeled with 2 nonbridge and 2 bridge 18O atoms
(denoted as 2,2), 7% contain 2 nonbridge and 1 bridge 18O atoms (2,1) and 7% contain 1
nonbridge and 2 nonbridge (1,2) 18O atoms. For the α-phosphoryl group, the 31P NMR
spectrum (Figure 2a) indicates that in the original β-[18O4]-ATP, 96% contains 1 bridge 18O
atoms (0,1) and 4% contain no label (0,0). The average 18O content of the 4 oxygen atoms
attached to the β-phosphoryl group is 96%. After 8 h, the fraction of the ATP with 2
nonbridge and 2 bridge 18O atoms at the β-phosphoryl group (2,2), relative to that fraction
of ATP with 2 nonbridge and 1 bridge 18O (2,1), has been reduced from 92% to 58%
(Figure 2d). For the α-phosphoryl group, the fraction of ATP with 1 bridge 18O atom (0,1),
relative to that with 1 nonbridge 18O atom has decreased from 100% to 38% (Figure 2b).
The rate of the PIX reaction (vPIX) was calculated using Eqn. 2 and the data for additional
time points are collected in Table 2. The average ratio of the PIX rate to the rate for the
loss of ATP (vPIX/vchem) is 1.6 ± 0.3. In the presence of 5 mM AMP, there was no measureable
increase in the rate of the PIX reaction under these conditions. The observation of the
positional isotope exchange reaction is consistent with the formation of a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
pyrophosphorylated enzyme in the absence of added L-glutamine.
19
ACS Paragon Plus Environment

Biochemistry
Page 20 of 30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. 31P NMR spectra of 18O-labeled ATP before and after the addition of Cj1418. (A)
Spectrum of the α-phosphoryl group of [18O4]-ATP (13) before the addition of Cj1418. (B)
Spectrum of the α-phosphoryl group of the ATP after 8 h of incubation with Cj1418. (C)
Spectrum of the β-phosphoryl group of [18O4]-ATP (13) before the addition of Cj1418. D. β-
phosphoryl group of the ATP after incubation with Cj1418 for 8 h. Additional details are
provided in the text. The first number in parentheses indicates the number of non-bridging
18O atoms attached to respective phosphoryl group in ATP and the second indicates the
number of bridging 18O atoms.
20
ACS Paragon Plus Environment

Page 21 of 30
Biochemistry
1
2
3
4
Table 2: Positional isotope exchange for the α- and β-phosphoryl groups with β-[18O4]-ATP.
5
6
7
8
9
Time % α-P % α-P % β-P % β-P
F
ATP
(mM)
5.00
X
ʋex
ʋchem
ʋex/ʋChem
(h)
0
(0,1)
100
86
(1,0)
0
(2,2)
92
(2,1)
8
(mM hr-1) (mM hr-1)
1
14
25
38
49
52
82
18
32
42
47
52
0.19 4.41 0.11
0.39 3.91 0.21
0.57 3.03 0.39
0.69 2.21 0.55
0.76 1.66 0.66
0.99
0.55
0.41
0.33
0.29
0.59
0.27
0.25
0.23
0.22
1.68
2.04
1.64
1.43
1.32
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
75
68
8
62
58
12
15
51
53
48
48
Detection of Phosphorylated Intermediate. In Scheme 2 we have proposed that
the pyrophosphorylated enzyme (Enz-X-Pβ-Pγ) is hydrolyzed to generate a phosphorylated
enzyme intermediate (Enz-X-Pβ) and in the last step this intermediate is utilized to
phosphorylate L-glutamine. If this last step is reversible then a molecular isotope exchange
reaction (MIX) can be used to provide experimental support for the formation of Enz-X-Pβ.
This exchange reaction is illustrated in Scheme 7. In the MIX experiment, Cj1418 was
incubated with 10 mM [15N-amide]-
L-glutamine (21) and 10 mM of unlabeled L-glutamine
phosphate (2) in the absence of added ATP. The products of the reaction mixture were
examined by both 31P and 15N NMR spectroscopy after a period of 12 h. In Figure 3a is the
31P NMR spectrum of the
L
-glutamine-P (2) prior to the addition of Cj1418 and the [15N-
amide]- -glutamine (21). Two resonances are observed at -3.56 and -3.91 ppm,
L
representing the syn and anti-conformations of the product. After the addition of Cj1418
and [15N-amide]-
-glutamine, four new resonances are observed, representing L-glutamine-
L
P where the two phosphorus resonances are now coupled to 15N (compound 22). In the
corresponding 15N NMR spectra (Figure 3c), a single 15N-resonance is observed at 111.92
ppm for the amide nitrogen of the labeled
L
-glutamine (21). After incubation with L-
21
ACS Paragon Plus Environment

Biochemistry
Page 22 of 30
1
2
3
4
glutamine-P and Cj1418, four new resonances are observed from 146.06 and 142.18 ppm.
5
6
These resonances represent the syn and anti-conformations of the L-glutamine-P products
7
8
9
and coupling with 31P of 19 Hz.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 7: Molecular isotope exchange reaction for Cj1418.
Figure 3. 31P and 15N NMR spectra for the MIX reaction with Cj1418. (A) 31P NMR
spectrum of
-glutamine phosphate. (B) 31P NMR spectrum after 10 μM Cj1418 was
incubated with 10 mM -glutamine
-glutamine phosphate (2) and 10 mM [15N-amide]-
(21) for 12 h. (C) 15N NMR spectrum of [15N-amide]- -glutamine (21). (D) 15N NMR
spectrum after 10 µM Cj1418 was incubated with 20 mM [15N-amide]-
-glutamine and 20
mM -glutamine phosphate (2) for 12 h.
L
L
L
L
L
L
22
ACS Paragon Plus Environment

Page 23 of 30
Biochemistry
1
2
3
4
5
6
DISCUSSION
Mechanism of Action. The proposed mechanism of action for
L
-glutamine kinase is
7
8
9
presented in Scheme 2. In this mechanism a nucleophile in the active site first attacks the
β-phosphoryl group of ATP to form a pyrophosphorylated enzyme intermediate (Enz-X-Pβ-
Pγ) and AMP. The pyrophosphorylated intermediate is subsequently hydrolyzed to
generate a phosphorylated intermediate (Enz-X-Pβ) and Pi. In the last step the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
phosphorylated intermediate is used to phosphorylate L-glutamine on the amide nitrogen.
Utilization of amino acid sequence alignments with the closest functional homologues to
Cj1418 and site directed mutagenesis experiments, we have demonstrated that His-737 is
the most likely residue in the active site of this enzyme to function as the primary
nucleophile.
In the proposed mechanism, the γ-phosphoryl group of ATP is converted to
phosphate and the β-phosphoryl group is ultimately used to phosphorylate L-glutamine.
An alternative mechanism could have been written with the opposite outcome for these
two phosphoryl groups of ATP. However, we have now demonstrated using β-[18O4]-ATP
that the β-phosphoryl group is exclusively utilized to phosphorylate L-glutamine. This
result was confirmed by conducting the Cj1418-catalyzed reaction in [18O]-H2O, where the
only labeled product was inorganic phosphate.
Positional isotope exchange (PIX) and molecular isotope exchange (MIX)
experiments were used to further support the formation of the pyrophosphorylated (Enz-
X-Pβ-Pγ) and phosphorylated enzyme intermediates (Enz-X-Pβ) in the overall reaction
mechanism. In the PIX experiment, β-[18O4]-ATP was incubated with the enzyme in the
absence of
L
-glutamine in an attempt to observe the migration of the α/β-bridge oxygen to
23
ACS Paragon Plus Environment

Biochemistry
Page 24 of 30
1
2
3
4
the α-nonbridge positions in ATP (Scheme 6). As conducted, the PIX reaction can only
5
6
occur if the enzyme is pyrophosphorylated by ATP in the absence of L-glutamine and this
7
8
9
reaction must be reversible. In essence, the experiment measures the partitioning of the
[Enz-X-Pβ-Pγ ꢀ AMP] complex. When this complex reverts back to Enz-X and ATP, a PIX
reaction will be observed if the α-phosphoryl group of AMP is able to rotate in the active
site. The PIX rate will then be governed by the rate at which the ATP can be reformed and
dissociate from the active site into solution.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The partitioning forward is potentially governed by two related events. The AMP
may dissociate from the active site or the pyrophosphorylated enzyme may be hydrolyzed
to the phosphorylated enzyme. The dissociation of AMP is essentially irreversible since the
initial concentration of AMP is equivalent to the enzyme concentration used in this
experiment (10 μM). We attempted to enhance the PIX rate by adding unlabeled AMP to
the reaction mixture.20 If the partitioning forward was governed by AMP dissociation, then
increasing the concentration of AMP would enhance the steady-state concentration of the
[Enz-X-Pβ-Pα ꢀ AMP] complex and the overall PIX rate would increase. However, the
addition of AMP had no effect on the PIX rate and no ATP was directly formed from the
added unlabeled AMP. This result indicates that the partitioning forward is actually
governed by the rate at which the [Enz-X-Pβ-Pγ ꢀ AMP] complex is hydrolyzed to [Enz-X-Pβ ꢀ
Pγ]. Thus, the experimentally determined partitioning ratio (vPIX/vchem) of 1.6 for the [Enz-
X-Pβ-Pα ꢀ AMP] complex indicates that ATP is reformed and dissociates from the active site
1.6 times faster than this complex is hydrolyzed to irreversibly form [Enz-X-PβꢀPi ꢀ AMP].
It should also be noted that the average rate constant for the PIX reaction (obtained
from the average value for vex in Table 2) is relatively small at ~54 h-1 (0.54 mM h-1/0.01
24
ACS Paragon Plus Environment

Page 25 of 30
Biochemistry
1
2
3
4
5
6
7
8
9
mM enzyme). Since the dissociation of AMP and the hydrolysis of the pyrophosphorylated
enzyme intermediate are essentially irreversible, the enzyme must somehow be recycled
back to free enzyme. This can only occur via the hydrolysis of the [Enz-X-Pβ] complex to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[Enz-X]. The rate constant for the net loss of ATP in the absence of
from the average value of vchem in Table 2) is 31 h-1 (0.31 mM h-1/0.01 mM enzyme). The
kcat for the overall reaction (2.5 s-1) in the presence of -glutamine is 2.9 x 104 fold faster
L-glutamine (obtained
L
than this value. This clearly indicates that L-glutamine is much better than water in
reacting with the [Enz-X-Pβ] intermediate. PIX experiments have previously been used to
characterize the pyrophosphorylated enzyme intermediate formed in the reaction
catalyzed by pyruvate phosphate dikinase.20
Experimental support for the [Enz-X-Pβ] intermediate was obtained with the MIX
experiment where free enzyme (Enz-X) was phosphorylated with L-glutamine phosphate in
the absence of any added nucleotide (Scheme 7). The phosphorylated enzyme
intermediate was subsequently captured by the 15N-labeled
15N-labeled
L
-glutamine to generate the
L
-glutamine phosphate (22). This exchange reaction can only occur with the
formation of the [Enz-X-P] intermediate.
Substrate Specificity. We have identified four additional substrates for Cj1418 in
addition to -glutamine. These new substrates include the hydroxamate derivatives of
glutamate (7) and -aspartate (9), the hydrazide of -glutamate (8), and -glutamine (6).
Other compounds were tested as alternative substrates such as -glutamate and
asparagine, however neither of these compounds exhibited any detectable activity.
L
L-
L
L
D
L
L-
L
-
Glutamine derivatives with either the α-amino or α-carboxy groups removed were not
25
ACS Paragon Plus Environment

Biochemistry
Page 26 of 30
1
2
3
4
substrates, however,
D
-glutamine was shown to be a substrate and thus the
5
6
stereochemistry at the α-carbon is not vital.
7
8
9
The second best substrate for Cj1418 is γ-glutamyl hydroxamate (7) and formation
of an unstable O-phosphorylated derivative (15). Previously, compound 15 has been
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
shown to be an inhibitor of γ-L-glutamyl-L-cysteine synthetase where a highly reactive
isocyanate intermediate was proposed via a Lossen rearrangement.27 With Cj1418 we did
not obtain any evidence for the formation of an isocyanate derivative from 7. However, the
13C and 31P NMR spectra clearly indicate the formation of pyroglutamic acid (16) and the
hydroxyl amine O-phosphate ester (14). In contrast, the phosphorylated derivative of β-
aspartyl hydroxamate (18) appears to be relatively stable under the reaction conditions.
The hydrazide derivative of
unstable phosphorylated product (18). The final products were determined to be
phosphate and -pyroglutamate (16). The formation of phosphate, presumably arises from
L-glutamate (9) is also a substrate that produces a relatively
L
the nonenzymatic hydrolysis of the hydrazide derivative of phosphate. However, this
intermediate was not observed directly in the 31P NMR spectrum of the reaction mixture.
Conclusions. Here we have determined the substrate profile for L-glutamine
kinase, an enzyme that functions in the formation of the O-methyl phosphoramidate
modification in the capsular polysaccharide in C. jejuni. We have proposed a chemical
reaction mechanism for the phosphorylation of L-glutamine and have provided kinetic
evidence for the formation of pyrophosphorylated and phosphorylated enzyme
intermediates in this transformation.
26
ACS Paragon Plus Environment

Page 27 of 30
Biochemistry
1
2
3
4
ꢁ ASSOCIATED CONTENT
5
6
7
Supporting Information
8
9
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢁ AUTHOR INFORMATION
Corresponding Author
*raushel@tamu.edu
ORCID
Frank M. Raushel: 0000-0002-5918-3089
Notes
The authors declare no competing financial interest.
ꢁ ACKNOWLEDGMENTS
This work was supported in part by grants from the National Institutes of Health
(GM 122825).
27
ACS Paragon Plus Environment

Biochemistry
Page 28 of 30
1
2
3
4
ꢁ REFERENCES
5
6
7
8
9
[1] Young, K. T., Davis, L. M., and Dirita, V. J. (2007) Campylobacter jejuni: molecular biology and
pathogenesis, Nat. Rev. Microbiol. 5, 665-679.
[2] Roberts, I. S. (1996) The biochemistry and genetics of capsular polysaccharide production in
bacteria, Annu. Rev. Microbiol. 50, 285-315.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[3] Karlyshev, A. V., Champion, O. L., Churcher, C., Brisson, J.-R., Jarrell, H. C., Gilbert, M., Brochu, D.,
St Michael, F., Li, J., Wakarchuk, W. W., Goodhead, I., Sanders, M., Stevens, K., White, B.,
Parkhill, J., Wren, B. W., and Szymanski, C. M. (2005) Analysis of Campylobacter jejuni
capsular loci reveals multiple mechanisms for the generation of structural diversity and the
ability to form complex heptoses, Mol. Microbiol. 55, 90-103.
[4] St., M. F., M., S. C., Jianjun, L., H., C. K., Huan, K. N., Suzon, L., W., W. W., Jean-Robert, B., and A., M.
M. (2002) The structures of the lipooligosaccharide and capsule polysaccharide of
Campylobacter jejuni genome sequenced strain NCTC 11168, Eur. J. Biochem. 269, 5119-
5136.
[5] McNally, D. J., Lamoureux, M. P., Karlyshev, A. V., Fiori, L. M., Li, J., Thacker, G., Coleman, R. A.,
Khieu, N. H., Wren, B. W., Brisson, J.-R., Jarrell, H. C., and Szymanski, C. M. (2007)
Commonality and Biosynthesis of the O-Methyl Phosphoramidate Capsule Modification in
Campylobacter jejuni, J. Biol. Chem. 282, 28566-28576.
[6] Karlyshev, A. V., Linton, D., Gregson, N. A., Lastovica, A. J., and Wren, B. W. (2000) Genetic and
biochemical evidence of a Campylobacter jejuni capsular polysaccharide that accounts for
Penner serotype specificity, Mol. Microbiol. 35, 529-541.
[7] Taylor, Z. W., Brown, H. A., Holden, H. M., and Raushel, F. M. (2017) Biosynthesis of Nucleoside
Diphosphoramidates in Campylobacter jejuni, Biochemistry 56, 6079-6082.
[8] Taylor, Z. W., Brown, H. A., Narindoshvili, T., Wenzel, C. Q., Szymanski, C. M., Holden, H. M., and
Raushel, F. M. (2017) Discovery of a Glutamine Kinase Required for the Biosynthesis of the
O-Methyl Phosphoramidate Modifications Found in the Capsular Polysaccharides of
Campylobacter jejuni, J. Am. Chem. Soc. 139, 9463-9466.
[9] Taylor, Z. W., and Raushel, F. M. (2018) Cytidine Diphosphoramidate Kinase: An Enzyme
Required for the Biosynthesis of the O-Methyl Phosphoramidate Modification in the
Capsular Polysaccharides of Campylobacter jejuni, Biochemistry 57, 2238-2244.
[10] Morrison, J. F., Griffiths, D. E., and Ennor, A. H. (1957) The purification and properties of
arginine phosphokinase, Biochem. J. 65, 143-153.
[11] Ennor, A. H., Rosenberg, H., and Armstrong, M. D. (1955) Specificity of Creatine Phosphokinase,
Nature 175, 120.
[12] Roush, R. F., Nolan, E. M., Löhr, F., and Walsh, C. T. (2008) Maturation of an Escherichia coli
Ribosomal Peptide Antibiotic by ATP-Consuming N−P Bond Formation in Microcin C7, J. Am.
Chem. Soc. 130, 3603-3609.
[13] Cooper, R. A., and Kornberg, H. L. (1967) The mechanism of the phosphoenolpyruvate
synthase reaction, Biochimica et Biophysica Acta (BBA) - General Subjects 141, 211-213.
[14] Evans, H. J., and Wood, H. G. (1968) The mechanism of the pyruvate, phosphate dikinase
reaction, Proceedings of the National Academy of Sciences 61, 1448-1453.
[15] Stogios, P. J., Cox, G., Spanogiannopoulos, P., Pillon, M. C., Waglechner, N., Skarina, T., Koteva, K.,
Guarné, A., Savchenko, A., and Wright, G. D. (2016) Rifampin phosphotransferase is an
unusual antibiotic resistance kinase, Nat. Commun. 7, 11343.
[16] Qi, X., Lin, W., Ma, M., Wang, C., He, Y., He, N., Gao, J., Zhou, H., Xiao, Y., Wang, Y., and Zhang, P.
(2016) Structural basis of rifampin inactivation by rifampin phosphotransferase,
Proceedings of the National Academy of Sciences 113, 3803-3808.
28
ACS Paragon Plus Environment

Page 29 of 30
Biochemistry
1
2
3
4
5
6
7
8
9
[17] Finn, R. D., Attwood, T. K., Babbitt, P. C., Bateman, A., Bork, P., Bridge, A. J., Chang, H.-Y.,
Dosztányi, Z., El-Gebali, S., Fraser, M., Gough, J., Haft, D., Holliday, G. L., Huang, H., Huang, X.,
Letunic, I., Lopez, R., Lu, S., Marchler-Bauer, A., Mi, H., Mistry, J., Natale, D. A., Necci, M., Nuka,
G., Orengo, C. A., Park, Y., Pesseat, S., Piovesan, D., Potter, S. C., Rawlings, N. D., Redaschi, N.,
Richardson, L., Rivoire, C., Sangrador-Vegas, A., Sigrist, C., Sillitoe, I., Smithers, B., Squizzato,
S., Sutton, G., Thanki, N., Thomas, P. D., Tosatto, Silvio C. E., Wu, C. H., Xenarios, I., Yeh, L.-S.,
Young, S.-Y., and Mitchell, A. L. (2017) InterPro in 2017—beyond protein family and domain
annotations, Nucleic Acids Res. 45, D190-D199.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[18] Lim, K., Read, R. J., Chen, C. C. H., Tempczyk, A., Wei, M., Ye, D., Wu, C., Dunaway-Mariano, D., and
Herzberg, O. (2007) Swiveling Domain Mechanism in Pyruvate Phosphate Dikinase,
Biochemistry 46, 14845-14853.
[19] Rose, I. A. (1979) Positional Isotope Exchange Studies of Enzyme Mechanisms, In Adv. Enzymol.
50, 361.
[20] Wang, H. C., Ciskanik, L., Dunaway-Mariano, D., Von der Saal, W., and Villafranca, J. J. (1988)
Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase,
Biochemistry 27, 625-633.
[21] Melby, E. S., Soldat, D. J., and Barak, P. (2011) Synthesis and Detection of Oxygen-18 Labeled
Phosphate, PLoS One 6, e18420.
[22] Wehrli, W., Verheyden, D., and Moffatt, J. (1965) Dismutation Reactions of Nucleoside
Polyphosphates. II. Specific Chemical Syntheses of α-, β-, and γ-P32-Nucleoside 5'-
Triphosphates1, J. Am. Chem. Soc. 87, 2265-2277.
[23] Midelfort, C. F., and Rose, I. A. (1976) A stereochemical method for detection of ATP terminal
phosphate transfer in enzymatic reactions. Glutamine synthetase, J. Biol. Chem. 251, 5881-
5887.
[24] Cottam, G. L., Kupiecki, F. P., and Coon, M. J. (1968) A Study of the Mechanism of O-
Phosphorylhydroxylamine Synthesis Catalyzed by Pyruvate Kinase, J. Biol. Chem. 243, 1630-
1637.
[25] Litwin, S., and Wimmer, M. J. (1979) Correction of scrambling rate calculation for loss of
substrate, J. Biol. Chem. 254, 1859.
[26] Cohn, M., and Hu, A. (1978) Isotopic ((18)O) shift in (31)P nuclear magnetic resonance applied
to a study of enzyme-catalyzed phosphate—phosphate exchange and phosphate (oxygen)—
water exchange reactions, Proc. Natl. Acad. Sci. U. S. A. 75, 200-203.
[27] Katoh, M., Hiratake, J., and Oda, J. i. (1998) ATP-Dependent Inactivation of Escherichia coli γ-
Glutamylcysteine Synthetase by L-Glutamic Acid γ-Monohydroxamate, Biosci., Biotechnol.,
Biochem. 62, 1455-1457.
29
ACS Paragon Plus Environment