Tài liệu Báo cáo Y học: Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis The role of the allosteric activator GTP in coupling between glutamine hydrolysis and CTP synthesis potx

Steady-state kinetics of the glutaminase reaction of CTP synthaseThe role of the allosteric activator GTP in coupling between glutamine hydrolysis and CTP synthesis Martin Willemoe¨s1and

Steady-state kinetics of the glutaminase reaction of CTP synthase

The role of the allosteric activator GTP in coupling between glutamine hydrolysis and CTP synthesis

Martin Willemoe¨s1and Bent W Sigurskjold2

1

Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Copenhagen, Denmark;

2

Department of Biochemistry, August Krogh Institute, University of Copenhagen, Copenhagen, Denmark

CTP synthase catalyzes the reaction glutamine + UTP

+ ATP fi glutamate + CTP + ADP + Pi The rate

of the reaction is greatly enhanced by the allosteric activator

GTP We have studied the glutaminase half-reaction of CTP

synthase from Lactococcus lactis and its response to the

allosteric activator GTP and nucleotides that bind to

the active site In contrast to what has been found for the

Escherichia coli enzyme, GTP activation of the L lactis

enzyme did not result in similar kcatvalues for the

glutami-nase activity and glutamine hydrolysis coupled to CTP

synthesis GTP activation of the glutaminase reaction never

reached the levels of GTP-activated CTP synthesis, not even

when the active site was saturated with UTP and the

non-hydrolyzeable ATP-binding analog adenosine

5¢-[c-thio]tri-phosphate Furthermore, under conditions where the rate of

glutamine hydrolysis exceeded that of CTP synthesis, GTP

would stimulate CTP synthesis These results indicate that the L lactis enzyme differs significantly from the E coli enzyme For the E coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis

to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis An alternative mechanism for the action of GTP

on L lactis CTP synthase is suggested Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself

Keywords: CTP synthase; isothermal titration calorimetry; glutaminase activity; allosteric regulation; Lactococcus lactis

CTP synthase (EC 6.4.3.2) catalyzes the synthesis of CTP

from UTP by amination of the pyrimidine ring at the

4-position The enzyme has three functionally distinct sites;

the glutaminase site where glutamine hydrolysis occurs, the

active site where CTP synthesis takes place and the allosteric

site that binds GTP The reaction is thought to proceed via

phosphorylation of UTP by ATP to give an activated

intermediate 4-phosphoryl UTP and ADP [1,2] Ammonia

then reacts with this intermediate yielding CTP and Pias

illustrated in Scheme 1A Ammonia can either be utilized

directly or be generated from the hydrolysis of glutamine in

a GTP-activated reaction [3,4] Similar mechanisms to that

shown in Scheme 1A, have been shown now for several

amido transferase enzymes [5–7] Here the binding of an

already activated substrate, or activation of the substrate on

the enzyme, in this case by phosphorylation, precedes amination The overall reaction is as follows:

*

PPPrib O HN

N

OPO 3

2-PPPrib

O

O HN

N ATP NH3

PPPrib

H 2 N

O HN

N

OPO 3

2-PPPrib

NH 2

O N

N

A

PPPrib

O

O HN

N

PPPrib

H 2 N

O HN

N OPO 3

2-PPPrib

NH 2

O N

N

B

*

NH 3 ATP

PPPrib

H 2 N

O HN

N OH

Scheme 1 Proposed mechanisms of CTP synthesis The box indicates

an expected transition state like structure * indicates that the amino donor can either be free ammonia or ammonia generated from hydrolysis of glutamine.

Department of Chemistry, University of Copenhagen,

Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.

Fax: + 45 35320299, Tel.: + 45 35320239,

E-mail: martin@ccs.ki.ku.dk

Abbreviations: ADPNP, adenosine 5¢-[b,c-imido]triphosphate;

ATP-cS, adenosine 5¢-[c-thio]triphosphate; CPS, carbamoyl

phosphate synthase; DON, 6-diazo-5-oxo-norleucine; GDH,

glutamate dehydrogenase; ITC, isothermal titration calorimetry.

Enzymes: CTP synthase (EC 6.4.3.2).

(Received 24 May 2002, revised 29 July 2002, accepted 9 August 2002)

ATPþ UTP þ glutamine ! ADP þ Piþ CTP

þ glutamate CTP synthase from Escherichia coli has been shown to

perform the following two partial reactions:

glutamine ! glutamate þ NH3 ½8 and

where the latter reaction takes place only in the presence

of UTP [1] Levitzki and Koshland originally suggested

a mechanism as shown in Scheme 1B [9] This

mechan-ism involves another intermediate, a carbinol amine,

formed prior to the phosphorylation step It was found

that the E coli enzyme would hydrolyze glutamine at a

steady-state rate similar to the rate of GTP-activated

CTP synthesis, if the enzyme was incubated in the

presence of GTP, UTP and the nonhydrolyzable ATP

analog ADPNP [8] Furthermore, it was shown that the

fold activation of the glutaminase activity by GTP was

similar to that of the overall CTP synthesis reaction It

was concluded that the effect of GTP was mainly to

enhance the rate of chemical steps of the glutaminase

reaction The finding that the kcat value was similar for

glutaminase activity in the presence of GTP, UTP and

ADPNP and CTP synthesis with ATP replacing

ADP-NP seems in agreement with the mechanism in

Scheme 1B, but maybe less so with the mechanism in

Scheme 1A

We have previously characterized the CTP synthase from

Lactococcus lactis[4] This enzyme appears to be a more

stable tetramer than the E coli [10], yeast [11] and

mammalian [12,13] enzymes that all require the presence

of UTP and/or ATP to form tetramers Therefore, the

L lactis CTP synthase is an attractive candidate for

mechanistic and structure–function studies since equilibria

between different oligomeric forms of the enzyme will not

interfere with the interpretation of the data

In this research, we have analyzed the steady state

kinetics of the glutaminase reaction of CTP synthase from

L lactis in order to distinguish between the effects of

GTP on the glutaminase reaction and the CTP synthesis

reaction The results from this work suggests that there

are major differences between E coli and L lactis CTP

synthase with respect to the regulation of glutamine

hydrolysis

E X P E R I M E N T A L P R O C E D U R E S

Materials

Bovine GDH, nucleotides, and all other chemicals were

obtained from Sigma, except ATP-cS which was obtained

from Roche CTP synthase from L lactis was purified as

described previously [4] 6-Diazo-5-oxo-norleucine

(DON)-labeled CTP synthase was obtained by incubating overnight

at 25C, with 100 lL of 8 mgÆmL)1of enzyme in 50 mM

Hepes, pH 8.0, 2 mMdithiothreitol and 5 mMDON The

protein concentration was determined by the bicinchoninic

acid procedure with reagents provided by Pierce Chemical

Company and with bovine serum albumin as a standard

The enzyme concentration was calculated using an Mrof

60 000 per subunit [4]

Spectrophotometric assay of CTP synthesis and glutaminase activity

Assays were performed at 30C in 50 mMHepes, pH 8.0,

2 mMdithiothreitol For CTP synthesis, the conversion of UTP to CTP with De291¼ 1338 cm)1ÆM )1was measured as described previously [4] For glutaminase activity, a con-tinuous coupled assay was used where the glutamate produced by CTP synthase was oxidized by GDH and monitored by the reduction of NAD+ to NADH with

De340¼ 6300 cm)1ÆM )1as described previously [14] Unless otherwise stated, the MgCl2concentration was 20 mM, the glutamine concentration was 15 mM, and the concentration

of each nucleotide when present in the assay was 1 mM

Isothermal titration calorimetry (ITC) assay

of glutaminase activity and CTP synthesis CTP synthase at concentrations between 0.029 and 1.16 lM

was loaded in the reaction cell of an MCS active temperature compensation isothermal titration calorimeter from Micro-Cal, LLC (Northampton, MA, USA) The steady-state heat evolvement from successive injections of glutamine or GTP was recorded as a displacement of the baseline as illustrated

in Fig 1A The power value of the baseline and its

0 2 4 6 8 10 12 14 30

32 34

Time (min)

-1 )

A

B

60 70 80 90 100 110 120 130 140 150 30

31 32 33

Time (min)

-1 )

Fig 1 Isothermal titration calorimetry of the glutaminase activity of

L lactis CTP synthase (A) Enthalpogram showing the recording of steady-state rates for the hydrolysis of glutamine at increasing substrate concentrations measured as the displacement of the baseline The peaks observed at each injection time are derived from the heat of dilution

of glutamine into the reaction cell (B) The heat generated by hydrolysis

displacement with each injection was directly obtained from

the data files as the value recorded just prior to the

subsequent injections Dividing the power with the molar

reaction enthalpy gives the steady-state rate The assay

conditions were as described above for the

spectrophoto-metric assays The molar reaction enthalpy, DH, of

gluta-mine hydrolysis or CTP synthesis under the experimental

conditions as outlined above, was determined by recording

the complete hydrolysis of between 0.05 and 0.15 lmol of

glutamine injected into the reaction cell containing between

1.7 and 17 lMCTP synthase and integrating the entire heat

evolvement over time For the measurement of DH for

CTP synthesis the enzyme was incubated with ATP, UTP,

GTP and MgCl2as described above Although hydrolysis

of ATP will take place under these conditions before and

after injection of glutamine and prior to kinetic

experi-ments, it will not interfere with the measurements as this

hydrolysis is slow and steady-state will prevail for at least

8 h, greatly exceeding the time required for the experiments

(about 20Ờ40 min) The values of DH were found to be

)29.7 ổ 0.8 kJẳmol)1 and )47.3 ổ 0.3 kJẳmol)1 for the

glutaminase reaction and the CTP synthesis reaction,

respectively The first value agrees well with the value of

Kishore et al [15]

The steady-state rate, or initial velocity, vj for each

injection, j, was determined from

vjỬ DPj

where DP is the change in compensation power of the

calorimeter necessary to maintain the temperature in the

reaction cell upon injection of substrate This is represented

by a shift in the baseline position DH is the molar enthalpy

of the reaction under the chosen experimental conditions,

and [E] is the enzyme concentration The correction for the

enzyme dilution and liquid displacement upon substrate

injection was calculated from

ơEjỬ ơEj1exp Vinj

Vcell

đ2ỡ where Vinjand Vcellare the volumes of the injectant solution

and the reaction cell, respectively The corrections for

the dilution of substrate already present in the reaction

cell upon further substrate injection and for the decrease

in substrate concentration with time were calculated from

ơSjỬ ơSj1 exp Vinj

Vcell

 vj1tj1

ợ ơSsyr 1 exp Vinj

Vcell

where [S]jis the accumulated substrate concentration at the

time of injection j, [S]j)1is the substrate concentration at the

time of injection j)1, vj)1is the steady-state rate of enzyme

activity prior to injection j, tj)1is the time between injection

j)1 and j, [S]syris the concentration of the injectant in the

syringe

Analysis of initial velocity data

Analysis of saturation curves was performed by nonlinear

regression using

vỬkcatơEơS

where kcatis the turnover number for the enzyme, [S] is the substrate concentration and Km is the HenriỜMichaelisỜ Menten constant Partial inhibition of the glutaminase activity induced by GTP was analysed using a modification

of the equation by LiCata and Allewell [16]

vỬkcatơE ợ kcat;inhơEđơA=I0:5ỡ

n

1ợ KA=ơA ợ đơA=I0:5ỡn đ5ỡ where kcat,inhis the turnover number for the enzyme fully complexed with the activator A, with an activation constant,

Ka, that in turn shows cooperative partial inhibition with an inhibition constant for half-maximal inhibition, I0.5, and a Hill-coefficient, n Initial velocity data from the activation of CTP synthesis or glutaminase activity by GTP as measured spectrophotometrically was analysed using

vỬ kcat;1ơE ợkcat;2ơEơA

where kcat,1 and kcat,2 are the turnover numbers for the enzyme in the absence of or fully saturated with the activator, respectively GTP inhibition of the ammonia dependent CTP synthesis reaction for DON labeled CTP synthase was performed using

% InhibitionỬ 100% ơI

n

Kn

where KIis the concentration of inhibitor that gives rise to 50% inhibition by the inhibitor I and n is the Hill-coefficient The standard errors presented are those given by the computer program (ULTRAFITfor the Macintosh vs 3.0, BioSoft)

R E S U L T S Steady state kinetics using ITC The use of a calorimetric assay for the study of the L lactis CTP synthase proved very useful, since a complete satura-tion curve for glutamine or GTP could be recorded quickly and reproducibly without complicating interference from the presence of added ligands (e.g the GTP absorbance at

291 nm that prevents the use of higher concentrations than about 0.3 mMGTP in the assay) A calorimetric assay has been shown recently to be generally applicable to most enzyme systems [17] However, the technique requires that

DH for the studied reaction is significantly different from zero Another condition that needs to be fulfilled in order to determine kcatfor an enzyme is that DH must be measured under experimental conditions in which complete turnover

of the substrate takes place Alternatively, one has to determine the equilibrium constant of the reaction to calculate DH Both the glutaminase reaction and the CTP synthesis reaction of CTP synthase are virtually irreversible and present no problem with respect to determining DH Figure 1A shows the raw calorimetric data (enthalpo-gram) obtained for glutamine hydrolysis by CTP synthase The complete hydrolysis of glutamine necessary for calcu-lating the molar enthalpy DH, and subsequently the rate of

the reaction, is shown in Fig 1B Finally, the calorimetric data converted to rate data can be fitted as conventional enzyme kinetic data From Fig 2 it can be seen that there is

an excellent agreement between measured initial velocities independently of the assay method used

Steady state kinetics of uncoupled-and CTP synthesis-coupled glutamine hydrolysis The ATP analogs, ATP-cS and ADPNP, did not serve as substrates (data not shown) but inhibited the CTP synthesis reaction (Fig 3) ADPNP, reported to inhibit the E coli enzyme with a Kisimilar to the dissociation constant for ATP [9], was a poor inhibitor of L lactis CTP synthase compared to ATP-cS On the basis of these results, ATP-cS was chosen as a binding analog of ATP CTP synthesis requires the presence of both the nucleotide substrates ATP and UTP When phosphorylation of UTP is hindered by the absence of ATP, only glutamine hydrolysis takes place GTP alone or in combination with UTP and ATP-cS gave a

Glutamine, mM

Glutamine, mM

A

B

0

0.05

0.1

0.15

0

1

2

3

ν, s-1

ν, s-1

Fig 2 Comparison of the ITC assay with spectrophotometric assays.

Initial velocities obtained from varying glutamine (A) Glutaminase

(circles) and CTP synthesis (squares) Open symbols represent data

reduction by GDH Closed symbols represent data obtained from

ITC (B) CTP synthesis in the absence (circles) and presence (squares)

spec-trophotometric measurement of UTP to CTP conversion Closed

symbols represent data obtained from ITC In panel (A) and (B) closed

(squares) and closed (circles), respectively, represent the same

data-points.

0 0.25 0.5 0.75 1 1.25

Inhibitor, mM

Fig 3 Inhibition of L lactis CTP synthase by ADPNP and ATP-cS CTP synthesis was measured spectrophotometrically as described in Experimental Procedures Inhibition was by ADPNP (circles) and by ATP-cS (triangles).

threefold or sixfold increase in kcat, respectively, compared

to the absence of nucleotides, whereas the Kmfor glutamine

was similar for all conditions (Table 1)

Adding ATP and UTP, so that CTP synthesis could take

place, gave a 1.7-fold increase in kcatwhen compared to

glutamine hydrolysis in the absence of nucleotides (Table 1)

The rate of CTP synthesis was dramatically influenced by

the presence of GTP, and 20 and 41-fold increases in kcat

were obtained with GTP concentrations of 0.1 mM and

1 mM, respectively However, only a modest decrease in Km

for glutamine was observed when compared to the absence

of GTP (Table 1)

Allosteric GTP activation of

uncoupled-and CTP synthesis-coupled glutamine hydrolysis

As was already indicated by the results in Table 1 and

discussed above, the kinetics of GTP activation of the

glutaminase half-reaction differed markedly on whether

the reaction was coupled to CTP synthesis or not (Fig 4A)

The glutaminase activity in the absence of GTP, represented

by kcat,1(Eqn 6) is not obtainable with the calorimetric assay

where GTP is varied, since the heat evolved representing this

activity is included in the baseline of the experiment

Therefore, the assay only measures the rate increase due to

the addition of GTP with a resulting kcatthat represents

kcat,2 (Eqn 6) When the ITC assay was used, the basal

glutaminase activity was therefore calculated for the

experimental conditions used for GTP activation on the

basis of the kinetic constants in Table 1, and those obtained

for the glutaminase reaction in the presence of 0.1 mMeach

of UTP and ATP-cS (data not shown) Apparently, the

value of Ka, kcat,1and kcat,2were similar regardless of the

active site being saturated with UTP and ATP-cS (1 mM

each) or not (0.1 mM each) (Table 2) However, GTP

concentrations above 1 mM appeared to partially inhibit

glutamine hydrolysis when UTP and ATP-cS were present

at only 0.1 mMeach This inhibition by GTP seemed to be

relieved by increasing the concentration of UTP and

ATP-cS to 1 mMeach (Fig 4B) In either case, as judged from

the values of kcat,1and kcat,2, the maximal GTP activation

of uncoupled glutamine hydrolysis was about 14-fold

(Table 2)

At UTP and ATP concentrations of 1 mMeach, a 49-fold

increase in kcatwas observed with a concomitant decrease in

Ka for GTP of about sevenfold compared to uncoupled

glutamine hydrolysis (Fig 4A and Table 2) GTP-activated

CTP synthesis in the presence of low concentrations

(0.1 mM each) of ATP and UTP showed a sevenfold

activation and a Kavalue three orders of magnitude lower

than for uncoupled glutamine hydrolysis where ATP-cS

replaced ATP (Fig 4C and Table 2)

In another experiment similar to that in Fig 4C, the GTP

activation of the glutaminase reaction and CTP synthesis

was compared (Fig 5) In the absence of GTP, the rate of

glutamine hydrolysis was significantly higher than the rate

of CTP synthesis so that the reactions appeared uncoupled

in terms of stochiometry However, GTP stimulated CTP

synthesis and apparently acted to coordinate or couple the

two reactions For comparison, the glutaminase activity

calculated from the kinetic constants of GTP activation in

the presence of 0.1 mMeach of ATP-cS and UTP (Table 2)

is indicated by the straight line in Fig 5

L lactisCTP synthase was incubated with the glutamine affinity analog, DON, that covalently labels an active site cysteine residue and thereby prevents the use of glutamine as amino donor for CTP synthesis [18] However, even though the enzyme had no detectable activity with glutamine, the

Fig 4 GTP activation of L lactis CTP synthase Data (A,B) were obtained by ITC, or by (C) the spectrophotometric CTP synthesis assay (A) GTP activation (squares) of CTP synthesis For comparison

shown within the same concentration range (B) GTP activation of the

(closed circles) each of UTP and ATP-cS (C) GTP activation of CTP

ammonia-dependent activity was fully retained, as was also

found for the E coli enzyme [9] GTP has previously been

reported to inhibit the NH4Cl-dependent CTP synthesis

reaction of the DON-labeled E coli enzyme, but not the

unmodified enzyme [8] This was also the case for the

L lactisenzyme (Fig 6) When this inhibition was analysed

as a function of the GTP concentration using Eqn 7 we

obtained a KI¼ 0.40 ± 0.05 and n ¼ 0.39 ± 0.02, results

that are very similar to those found for the E coli enzyme

[8] Interestingly, the inhibitory response to GTP binding in

this experiment shows negative cooperativity in contrast to

the activation experiments presented above, where

cooper-ativity is not observed

D I S C U S S I O N

The original model for the mechanism of GTP activation of

the E coli CTP synthase was rather complex, involving both

negative and positive cooperativity of GTP binding [8] However, we have not found cooperativity associated with GTP activation in these or previous studies [4] of the

L lactisenzyme Also, for the E coli enzyme, it appears that the cooperative phenomena that have been observed previously are only associated with equilibrium binding, but seem irrelevant in terms of kinetic activation by GTP [19] Since there appears to be no effect of GTP on the CTP synthesis reaction where NH4Cl is the amino donor, the activation by GTP seems exclusively associated with catalytic properties unique to the utilization of glutamine This observation also implies that there is no effect of GTP

on the rate of phosphorylation of UTP by CTP synthase, as

kcatfor CTP synthesis with NH4Cl as amino donor is similar

or higher than for the glutamine-dependent reaction satur-ated with GTP These observations, valid for both E coli [8] and L lactis CTP synthase [4], are important when dissecting the effect of GTP on glutamine-dependent CTP synthesis

As mentioned in the Introduction, the GTP activation of the E coli enzyme is proposed to be due mainly to an increase in the rate of glutamine turnover [8] In agreement with this proposal the fold activation by GTP for this enzyme is largely independent of the occupancy of the active site, i.e whether the ATP binding analog, ADPNP, and

Table 2 Kinetic constants for L lactis CTP synthase from varying GTP in the presence of glutamine and the indicated nucleotides.

a

Assays were performed as described in the legend to the indicated Fig Glutaminase refers to hydrolysis of glutamine without CTP

GTP, mM

0 10 20 30 40 50

Fig 6 GTP inhibition of DON-labeled L lactis CTP synthase The data were fitted to Eqn 7 and kinetic constants are given in the text.

GTP, mM

0

0.05

0.1

0.15

0.2

0.25

v, s-1

each of UTP and ATP Spectrophotometrical measurement of CTP

synthesis (squares) and glutaminase activity (circles) was performed as

described in Experimental procedures The solid line is calculated on

the basis of data (Table 2) from GTP activation of the glutaminase

shown for comparison.

UTP are present or not Also, it has been shown that the

E colienzyme in the presence of GTP, UTP and ADPNP

will hydrolyze glutamine with a kcat similar to CTP

synthesis, where ATP replaces ADPNP This latter

obser-vation suggests that the rate of glutaminase activity of the

E colienzyme is independent of the UTP-phosphorylation

reaction

The results presented here for the L lactis enzyme seem

to indicate significant mechanistic differences between this

enzyme and the E coli counterpart From Tables 1 and 2

it is seen that GTP would activate the uncoupled

glutaminase reaction, but not to the extent observed for

CTP synthesis Even though GTP activation of the

uncoupled glutaminase activity was clearly sensitive to

the occupation of the active site by the nucleotides UTP

and ATP-cS (Fig 4B), the kinetic constants deviated

significantly from those obtained for CTP synthesis under

similar conditions (Tables 1 and 2) Together, these results

seem to indicate that allosteric binding by GTP alone or in

combination with UTP and ATP-cS, is not capable of

activating glutamine hydrolysis in terms of kcatto the level

of CTP synthesis, where ATP replaced cS As

ATP-cS could not replace ATP in this coactivation with GTP,

we find it reasonable to suggest that the true coactivator is

the 4-phosphorylated UTP intermediate in a mechanism as

that of Scheme 1A

From Fig 4 and Table 2 it can be seen that the effect of

saturating the active site with ATP-cS and UTP was a relief

of partial inhibition by GTP at higher concentrations than

1 mM(Fig 4B) The exact mechanism behind this inhibition

cannot be resolved from our data, but apart from this

inhibition the kinetics were similar when ATP-cS and UTP

were present at 1 mM or 0.1 mM (Table 2) The results

presented in Fig 4B seem to exclude that the lower fold of

GTP activation of the uncoupled glutaminase reaction was

due to subsaturation with nucleotides binding to the active

site That CTP synthesis in the absence of GTP occurs with

a kcatthat is higher than for the glutaminase activity under

similar conditions, except that ATP-cS replaced ATP or in

the complete absence of nucleotides (Table 1), seems to

indicate an activation of the glutaminase reaction by the

substrate nucleotides alone A similar observation was made

with the E coli enzyme except that UTP and ADPNP also

activated the glutaminase reaction, though not to the same

extent as ATP and UTP [9] For the L lactis enzyme, a

plausible explanation may be that 4-phosphorylated UTP

by itself acts as a weak activator of glutamine hydrolysis

This activation is greatly enhanced by GTP binding to the

enzyme

From Table 2 it can be seen that the degree of saturation

with ATP and UTP, unlike ATP-cS and UTP, has a large

influence on Kafor GTP This correlation of a decrease in

Ka for GTP with the lowering of the concentration of

nucleotide substrates, has been reported previously [4]

Even though a full description of the mechanism of GTP

activation is not yet available, the difference in Kafor GTP

observed when ATP replaced ATP-cS appears to involve

structural changes exerted by formation of 4-phosphoryl

UTP, that in turn increases the affinity of the enzyme for

GTP

An interesting observation was that when ATP and

UTP were present at concentrations that give rise to CTP

synthesis at a rate lower than the rate of glutamine

hydrolysis, GTP still acted as an activator (Figs 4C and 5) One might have expected that if the role of GTP was solely to increase the rate of glutamine hydrolysis, there would have been no effect of adding GTP when the rate of CTP synthesis was limited by the concentration of nucleotide substrates, and not hydrolysis of glutamine In the narrow concentration interval from 0 to about 2 lM

GTP, the rate of CTP synthesis is stimulated 3–4-fold from

a level below to the level of uncoupled glutamine hydrolysis (Fig 5) This directly illustrates that GTP plays

G N

G N

A

C B

Fig 7 A working model for the structural movements in the L lactis CTP synthase monomer (A) uncoupled glutaminase activity, (B) CTP synthesis in the absence of GTP and (C) CTP synthesis in the presence

of GTP (A) Glutamine hydrolysis in the absence of nucleotides takes place on the enzyme without any large structural changes required (B) CTP synthesis in the absence of GTP only occurs at a slow rate due to

an equilibrium between the inactive and active form of the monomer with respect to CTP synthesis that involves rearrangements of the monomer that brings together the glutaminase site and the active site (C) GTP locks the enzyme in the active form for CTP synthesis and

activate the uncoupled glutaminase reaction the structure of the monomer represented by (C) must also include a minor but highly important rearrangement of sidechains in the active site in response to the formation of 4-phosphoryl UTP The ammonia dependent CTP synthesis could in this model proceed via an enzyme form similar to (A).

G, glutaminase site; N, CTP synthesis site.

an additional role in activation of L lactis CTP synthase

apart from stimulating the chemical steps of glutamine

hydrolysis

The results presented in Fig 6 further support a dual role

for GTP activation of CTP synthesis A mechanism in

which GTP only acts to increase the rate of hydrolysis of

glutamine has some flaws in explaining the inhibition of

DON-labeled enzyme when assayed with NH4Cl as amino

donor Therefore, Levitzki and Koshland suggested that

upon GTP binding, small structural changes took place in

the DON-labelled enzyme that also affected the active site in

an inhibitory manner [8] It may be that these structural

changes in the active site postulated by Levitzki and

Koshland are related to the GTP activation of CTP

synthesis at excess glutamine hydrolysis in the 0 to about

2 lMconcentration range of the activator in Fig 5 It seems

plausible that the active site gets shielded from the

environ-ment upon GTP binding to the DON-labelled enzyme in a

way so that ammonia can no longer enter the active site The

DON-labeled enzyme with GTP bound would then mimic

the active form of the enzyme with hydrolyzed glutamine in

the glutaminase site and nucleotide substrates in the active

site This is similar to the GTP-sensitive competitive

inhibition of NH4Cl utilization, exerted by glutamate

c-semialdehyde as found with the E coli enzyme [20]

Glutamate c-semialdehyde is an analog of glutamine that

mimics a tetrahedral reaction intermediate [20]

In our current model (Fig 7), GTP may act to close a

lid over the active site, a lid that in turn holds or rearranges

catalytically important residues, and residues that enable

the enzyme to perform a concerted glutamine hydrolysis

with the formation of 4-phosphoryl UTP Maybe GTP

could play a role in the formation of a tunnel for passing

ammonia from the glutaminase site to the active site Such

tunnels have been demonstrated in several glutamine

amidotransferases [5] Another enzyme, that also catalyzes

amino transfer from glutamine, is carbamoyl phosphate

synthase (CPS) which is the first enzyme of the de novo

pyrimidine biosynthesis For CPS, glutamine hydrolysis

has been shown to be greatly stimulated by

bicarbonate-dependent ATP hydrolysis, indicating that for this enzyme

the phosphorylated amino acceptor intermediate, carbonyl

phosphate, triggers an allosteric signal to the glutaminase

site [5] We imagine the same type of allosteric activation of

glutamine hydrolysis takes place by the phosphorylation of

UTP on CTP synthase, only that for CTP synthase this

allosteric effect exerted by the amino acceptor is strongly

controlled by GTP

A C K N O W L E D G E M E N T S

This work was supported by the Danish National Research

Foundation We gratefully acknowledge the expert technical

assist-ance by Dorthe Boelskifte We wish to express our gratitude to

Sine Larsen for support to M W and for comments to the

manuscript.

R E F E R E N C E S

1 von der Saal, W., Anderson, P.M & Villafranca, J.J (1985)

Mechanistic investigations of Escherichia coli

cytidine-5¢-triphos-phate synthetase Detection of an intermediate by positional

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