Conformational cycling in beta-phosphoglucomutase catalysis: reorientation of the beta-D-glucose 1,6-(Bis)phosphate intermediate.

Abstract

Activated Lactococcus lactis beta-phosphoglucomutase (betaPGM) catalyzes the conversion of beta-d-glucose 1-phosphate (betaG1P) derived from maltose to beta-d-glucose 6-phosphate (G6P). Activation requires Mg(2+) binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-)(1), K(m) = 49 +/- 4 microM for the substrate betaG1P and K(m) = 6.5 +/- 0.7 microM for the activator beta-d-glucose 1,6-bisphosphate (betaG1,6bisP). The observed transient accumulation of [(14)C]betaG1,6bisP (12% at approximately 0.1 s) in the single turnover reaction carried out with excess betaPGM (40 microM) and limiting [(14)C]betaG1P (5 microM) and betaG1,6bisP (5 microM) supported the role of betaG1,6bisP as a reaction intermediate in the conversion of the betaG1P to G6P. Single turnover reactions of [(14)C]betaG1,6bisP with excess betaPGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the betaG1,6bisP binds to the active site in two different orientations (one positioning the C(1)phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6)phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with betaPGM, [(14)C]betaG1P, and unlabeled betaG1,6bisP demonstrated complete exchange of label to the betaG1,6bisP during the catalytic cycle. Thus, the reorientation of the betaG1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of betaG1P suggest that the reorientation and phosphoryl transfer from betaG1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of betaG1,6bisP to betaG1P plus G6P was examined to evidence a significant stabilization of betaPGM aspartyl phosphate.

Full text

Page 1

Conformational Cycling in ?-Phosphoglucomutase Catalysis: Reorientation of the
?-D-Glucose 1,6-(Bis)phosphate Intermediate†
Jianying Dai,‡Liangbing Wang, Karen N. Allen,§Peter Radstrom,|and Debra Dunaway-Mariano*,‡
Department of Chemistry, UniVersity of New Mexico, Albuquerque, New Mexico 87131, Department of Physiology and
Biophysics, Boston UniVersity School of Medicine, Boston, Massachusetts 02118, and Department of Applied Microbiology,
Lund Institute of Technology, Lund UniVersity, P.O. Box 124, S-221 00 Lund, Sweden
ReceiVed January 20, 2006; ReVised Manuscript ReceiVed April 28, 2006
ABSTRACT: Activated Lactococcus lactis ?-phosphoglucomutase (?PGM) catalyzes the conversion of ?-D-
glucose 1-phosphate (?G1P) derived from maltose to ?-D-glucose 6-phosphate (G6P). Activation requires
Mg2+binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used
to define the steady-state kinetic constants kcat) 177 ( 9 s-1, Km) 49 ( 4 µM for the substrate ?G1P
and Km) 6.5 ( 0.7 µM for the activator ?-D-glucose 1,6-bisphosphate (?G1,6bisP). The observed transient
accumulation of [14C]?G1,6bisP (12% at ∼0.1 s) in the single turnover reaction carried out with excess
?PGM (40 µM) and limiting [14C]?G1P (5 µM) and ?G1,6bisP (5 µM) supported the role of ?G1,6bisP
as a reaction intermediate in the conversion of the ?G1P to G6P. Single turnover reactions of [14C]?G1,-
6bisP with excess ?PGM were carried out to demonstrate that phosphoryl transfer rather than ligand
binding is rate-limiting and to show that the ?G1,6bisP binds to the active site in two different orientations
(one positioning the C(1)phosphoryl group for reaction with Asp8, and the other orientation positioning
the C(6)phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover
reactions carried out with ?PGM, [14C]?G1P, and unlabeled ?G1,6bisP demonstrated complete exchange
of label to the ?G1,6bisP during the catalytic cycle. Thus, the reorientation of the ?G1,6bisP intermediate
that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the
opposite orientation. Published X-ray structures of ?G1P suggest that the reorientation and phosphoryl
transfer from ?G1,6bisP occur by conformational cycling of the enzyme between the active site open and
closed forms via cap domain movement. Last, the equilibrium ratio of ?G1,6bisP to ?G1P plus G6P was
examined to evidence a significant stabilization of ?PGM aspartyl phosphate.
Phosphoglucomutases catalyze the interconversion of
D-glucose 1-phosphate (G1P)1and D-glucose 6-phosphate
(G6P). Operating in the forward G6P-forming direction, this
reaction links polysaccharide phosphorolysis to glycolysis.
In the reverse direction, the reaction provides G1P for the
biosynthesis of exo-polysaccharides (2). There are two
classes of phosphoglucomutases, the R-phosphoglucomutases
(RPGM, EC 5.4.2.2), ubiquitous among eucaryotes and
procaryotes, and the ?-phosphoglucomutases (?PGM, EC
5.4.2.6), present in certain bacteria and protists. The two
classes of mutases are distinguished by their specificity for
R- and ?-D-glucose phosphates and by their protein-fold
family. The rabbit muscle RPGM (3) and the closely related
Pseudomonas aeruginosa RPGM/RPMM (4) are members
of the phosphohexomutase enzyme superfamily (5), whereas
?PGM (6) belongs to the haloalkanoic acid (HAD) enzyme
superfamily (7). The four-domain RPGM and RPGM/RPMM
(∼50 kDa) are approximately twice the size of the two-
domain ?PGM (∼25 kDa).
In RPGM (and in RPGM/RPMM), phosphoryl transfer is
mediated by an active site serine which forms a stable
phosphate ester linkage (half-life in water is ∼7 years) (8).
The catalytic cycle begins with the binding of RG1P to the
active site of the phosphorylated enzyme, followed by
phosphoryl transfer to the C(6)O (k ) 1000 s-1) (see Figure
1). The R-glucose 1,6-bisphosphate (RG16bisP) thus formed,
and tightly bound (8, 9, 11), must become reoriented in the
active site so that the C(1)phosphate can be transferred to
the active site nucleophile to yield the G6P product. It does
so by rotating 180° while still associated with the enzyme
(10).
In this paper, we examine the chemical pathway of the
Lactococcus lactis ?PGM-catalyzed conversion of ?G1P to
G6P.2This reaction is mediated by an active site aspartate
(Asp8), which forms an acyl phosphate as the covalent
enzyme intermediate (12). Kinetic methods were used to
demonstrate the intermediacy of ?G16bisP in G6P formation
†This work was supported by NIH Grant No. GM61099 to K.N.A
and D.D.-M.
* Corresponding author. Department of Chemistry, University of
New Mexico, Albuquerque, NM 87131. Tel: 505-277-3383. Fax: 505-
277-6202. E-mail: dd39@unm.edu.
‡University of New Mexico.
§Boston University School of Medicine.
|Lund University.
1Abbreviations: R-PGM, R-phosphoglucomutase; R-PGM/PMM,
duel specificity R-phosphoglucomutase/R-phosphomannomutase; ?PGM,
?-phosphoglucomutase; E, ?PGM-Mg2+; E-P, phospho-?-PGM-Mg2+;
?G1P, ?-D-glucose 1-phosphate; ?G16bisP, ?-D-glucose 1,6-(bis)-
phosphate; RG1P, R-D-glucose 1-phosphate; RG16bisP, R-D-glucose
1,6-(bis)phosphate; NADP, adenine dinucleotide 3′-phosphate; K+-
Hepes, potassium salt of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic
acid; HPLC, high-performance liquid chromatography.
7818
Biochemistry 2006, 45, 7818-7824
10.1021/bi060136v CCC: $33.50© 2006 American Chemical Society
Published on Web 06/03/2006

Page 2

and to show that the ?G16bisP intermediate is reoriented in
the active site by dissociation into solvent and then binding
in the opposite orientation. The rate and equilibrium for
phosphorylation of the active site Asp8 by ?G16bisP are
examined. A model for ?PGM catalysis is proposed that is
based on cycling of the enzyme between the open and closed
conformations observed in the reported ?PGM X-ray crystal
structures (6, 12-14).
MATERIALS AND METHODS
Enzymes and Reagents. R-D-[14C(U)]-glucose 1-phosphate
(specific activity (SA) ) 200 mCi/mmol) and D-[1-14C]-
glucose 6-phosphate (SA ) 49.3 mCi/mmol) were purchased
from Perkin-Elmer Life Sciences to serve as standards in
HPLC analysis. [14C(U)]maltose (SA ) 300 mCi/mmol) was
purchased from American Radiolabeled Chemicals, Inc. [14C-
(U)]?-D-glucose 1-phosphate and [14C(U)]?-D-glucose 1,6-
bisphosphate were prepared as described in Supporting
Information and reported in reference 15. Recombinant L.
lactis ?-phosphoglucomutase (?PGM) was prepared accord-
ing to a published procedure (16). Glucose 6-phosphate
dehydrogenase (EC 1.1.1.49) type IX from Baker’s yeast was
purchased from Sigma-Aldrich. G6P, ?G1P, NADP, and all
buffers were purchased from Sigma-Aldrich.
ActiVation by Phosphorylation. The steady-state kinetic
constants for ?PGM activation by phosphorylation were
measured by varying the concentration of the substrate ?G1P
between 0.5- and 5-fold Kmat fixed concentration of activator
?G1,6bisP (3, 5, 10, and 20 µM) in 50 mM K+Hepes (pH
7.0 and 25 °C) containing 0.001 µM ?PGM, 2 mM MgCl2,
0.2 mM NADP, and 3 unit/mL G6P dehydrogenase. The
formation of G6P was monitored by measuring the increase
in solution absorbance at 340 nm (? ) 6.2 mM-1cm-1)
resulting from the G6P dehydrogenase-catalyzed reduction
of NADP. Data were computer-fitted to eq 1. Where [A] is
the ?G1P concentration, [B] is the activator concentration,
V0is the initial velocity, Vmis the maximum velocity, KAis
the Michaelis constant for ?G1P, and KBis the Michaelis
constant for activator. The kcatwas calculated from the ratio
of Vmaxand the enzyme concentration.
Single TurnoVer Reactions. Single-turnover experiments
were performed at 25 °C using a rapid-quench instrument
from KinTek Instruments equipped with a thermostatically
controlled circulator. A typical experiment was carried out
by mixing 13 µL of buffer A (50 mM K+Hepes (pH 7.0)
and 2 mM MgCl2) containing ?PGM and 14 µL of buffer A
containing [14C(U)]?G1,6bisP or [1-14C]G6P plus ?G1,6bisP
or [14C(U)]?G1P plus ?G1,6bisP. The reaction was quenched
after a specified period of time with 193 µL of 1 M NaOH.
The quenched reaction mixture was passed through a 5-kDa
filter to remove the enzyme, and then loaded onto a Rainin
Dynamax high-performance liquid chromatography (HPLC)
system equipped with a CarboPac PA1 (Dionex) column (4
× 250 mm). The column was eluted at a flow rate of 1 mL/
min, first with 2 mL of solvent A (54 mM NaOH and 100
mM sodium acetate), then with a linear gradient (15 mL) of
solvent A to 53.7% solvent B (75 mM NaOH and 500 mM
sodium acetate), and last, with solvent B. Fractions (1 mL)
were collected, and their radioactivity was determined by
liquid scintillation counting. The retention times of ?G1P;
G6P; and ?G1,6bisP are 8, 15, and 21 min, respectively.
2No distinction between the R- and ?-anomers of glucose-6-
phosphate is offered because the solution epimerization of the G6P
C(1)OH is rapid (1).
FIGURE 1: The steps associated with phosphoglucomutase (E-X) activation by glucose 1,6-bisphosphate and the subsequent conversion of
glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P).
V0) Vm[A][B]/(KB[A] + KA[B] + [A][B]) (1)
?-Phosphoglucomutase Catalysis
Biochemistry, Vol. 45, No. 25, 2006 7819

Page 3

The radioactivity associated with the respective [14C]?G1P;
[14C]G6P; and [14C]?G1,6bisP fractions was used to calculate
the mole fraction of each species present in the reaction
mixture at termination. The observed rate constants for the
single turnover reactions were obtained by fitting the time
course data to the first-order eqs 2 and 3 using the computer
program Kaleidagraph. In these equations, k is the first-order
rate constant; [P]t and [S]t are the product and substrate
concentrations at time “t”, respectively; [S]maxis the initial
concentration of substrate; and [P]maxis the product concen-
tration at equilibrium.
RESULTS AND DISCUSSION
?-PGM ActiVation by Phosphorylation. To efficiently
catalyze the conversion of ?G1P to G6P, ?PGM must be
first phosphorylated at Asp8. In a previous study of L. lactis
?PGM, we showed that ?G1P can serve as the phosphoryl
group donor at a turnover rate of 0.8 s-1(12). Once formed,
the phosphorylated enzyme (E-P) catalyzes the phosphory-
lation of ?G1P to generate ?G1,6bisP. The ?G1,6bisP is
presumed to be the phosphoryl donor in subsequent turnover
reactions. In the present study of the L. lactis ?PGM,
activation by synthetic ?G1,6bisP was examined by using
initial velocity techniques.
Accordingly, the initial velocity of G6P formation was
measured as a function of ?G1P concentration at changing,
fixed ?G1,6bisP concentration. Because ?G1,6bisP and
?G1P bind to separate enzyme forms (E and E-P, respec-
tively), a parallel-pattern, double reciprocal plot, as is typical
of a Bi-Bi Ping-Pong kinetic mechanism (17), was observed
(Figure 2). Data fitting to the rate expression for the Bi-Bi
Ping-Pong kinetic mechanism defined a kcat) 177 ( 9 s-1,
Km) 49 ( 4 µM for the substrate ?G1P and Km) 6.5 (
0.7 µM for the activator ?G1,6bisP. The kcatvalue determined
at fixed saturating concentration of ?G1,6bisP and varied
concentration of ?G1P is however typically ∼100 s-1.
?PGM Binds ?G1,6bisP in Two Different Orientations.
The X-ray structure of the ?PGM complex of ?G1,6bisP is
depicted in Figure 3. In this structure, the enzyme assumes
a closed conformation with the cap domain bound to the
core domain, covering the active site and encapsulating the
bound ligand (Figure depicts the superposition of the
structure of the ?PGM(Mg2+)(?G1,6bisP) complex illustrat-
ing the closed conformation and the structure of the bound
phosphorylated ?PGM illustrating the open conformation).
The ?G1,6bisP C(6)phosphate interacts with electropositive
residues of the cap domain, and the C(1)phosphate is engaged
in covalent bond formation with the core domain Asp8
carboxylate. Thus, the structure provides a snapshot of the
enzyme as it moves along the reaction coordinate, in this
case one step beyond the Michaelis complex (see reaction
scheme in Figure 4A). The phosphorane intermediate shown
is formed from the bound ?G1,6bisP upon cap closure. We
presume that the stability of this reaction intermediate in the
crystal is the result of crystal packing forces stabilizing the
cap-closed conformation. In solution, the cap domain is free
to dissociate, thus, stimulating P-O bond cleavage in the
phosphorane to generate either the Michaelis complex of
phosphorylated enzyme (depicted in Figure 3A) with G6P
bound or the Michaelis complex of unphosphorylated enzyme
with ?G1,6bisP noncovalently bound.
For ?PGM catalysis in the ?G1P-forming direction, the
?G1,6bisP must be bound with the C(1)phosphate interacting
with the cap domain and the C(6)phosphate with the Asp8.
This orientation is termed E(?G1,6bisP)-1. For ?PGM
FIGURE 2: Plots of inverse velocity vs inverse ?G1P concentration
measured at various, fixed phosphoryl donor concentration of ?G1,-
6bisP, measured at 50 µM ?G1P in 50 mM K+Hepes buffer (pH
7.0) containing 2 mM MgCl2at 25 °C.
[P]t) [P]max(1 - e-kt)(2)
[S]t) [S]max- ([P]max(1 - e-kt)) (3)
FIGURE 3: (A) Superposition of the phospho-?PGM-Mg2+com-
plex illustrating the cap-open conformation (red; the phosphoryl
group is not shown) (6) and the ?PGM-Mg2+-6-phosphoglucose-
1-phosphorane complex (blue) illustrating the cap-closed conforma-
tion and the phosphorane intermediate (13). (B) Space filling model
of ?PGM in the open conformation as observed in the structure of
the phospho-?PGM-Mg2+complex. (C) Space filling model of
?PGM in the closed conformation as observed in the structure of
the ?PGM-Mg2+-6-phosphoglucose-1-phosphorane complex.
7820 Biochemistry, Vol. 45, No. 25, 2006
Dai et al.

Page 4

catalysis in the G6P-forming direction, the ?G1,6bisP must
be bound with the C(6)phosphate interacting with the cap
domain and the C(1)phosphate with the Asp8. This orienta-
tion (observed in the structure shown in Figure 3) is termed
E(?G1,6bisP)-2. To determine if one orientation is preferred
over the other, single turnover reactions between ?PGM and
limiting [14C]?G1,6bisP were carried out. As is illustrated
in Figure 4A, the collision between ?PGM and [14C]?G1,-
6bisP will form the Michaelis complexes E(?G1,6bisP)-1
and E(?G1,6bisP)-2. Cap closure will produce the corre-
sponding phosphorane intermediate (E(?G1,6bisP)-1* or
E(?G1,6bisP)-2*, which upon cap opening will revert to the
corresponding Michaelis complex or generate the corre-
sponding product complex E-P(?G1P) or E-P(?G6P). Once
formed, the product complexes will dissociate to E-P and
the product ligand. If the product ligand binds to the free
enzyme which is present in excess, the dead-end complex
E(?G1P) or E(?G6P) will be formed. The E-P can bind ?G1P
or ?G6P from the ligand pool and undergo the reverse
reaction sequence. In this manner, the reactions shown in
Figure 4A can attain equilibrium.
On the basis of a steady-state kcat ) 177 ( 9 s-1for
multiple turnover of ?G1P by the ?G1,6bisP-activated
enzyme, we anticipated that a single turnover reaction of
[14C]?G1,6bisP would be fast, perhaps too fast to monitor
by rapid quench. Nevertheless, the time courses for [14C]?G1P
and [14C]?G6P formation from the reaction of [14C]?G1,-
6bisP with excess ?PGM were measured to determine the
rate of phosphoryl transfer from[14C]?G1,6bisP to the Asp8
and to determine the ratio of [14C]?G1P and [14C]?G6P
formed as product. This ratio might reflect the relative rates
of E-P(?G1P) and E-P(?G6P) formation and, thus, the
preference of the enzyme for formation of and catalysis
within the Michaelis complexes E(?G1,6bisP)-1 and E(?G1,-
6bisP)-2.
The single turnover reaction was carried out using enzyme
in large excess to reactant (5 µM [14C]?G1,6bisP reacted
with 40 µM ?PGM) (Figure 4B) to ensure that the reactant
and products are enzyme-bound. For comparison, the reaction
was also carried with a 2:1 ratio of enzyme to reactant (10
µM [14C]?G1,6bisP with 20 µM ?PGM) (Figure 4C). At the
lower enzyme-to-ligand ratio, it is likely that the enzyme-
product complexes will release a significant amount of
product to solution. The time courses measured under two
sets of conditions show that both [14C]?G1P and [14C]?G6P
are formed in the reaction. Notably, the [14C]?G1P formation
appears to be completed within the first 4 ms time point, at
which point the ratio of [14C]?G6P to [14C]?G1P is ∼2:1.
The remainder of the time course reflects the approach to
equilibrium, which is different under the two sets of reaction
conditions. This difference can be attributed to the difference
in the ratio of the enzyme to ligand, which for the smaller
ratio translates into a greater contribution from solvent
equilibria of [14C]?G1P; [14C]?G1,6bisP; and [14C]G6P. The
time courses of Figure 4B,C are fitted with first-order rate
equations to define the kobs values reported in the figure
legends. However, we note that the kobsvalue for [14C]?G1P
formation is defined only by the 4 ms time point, and
therefore, the data define a minimum value as an estimate
for the kobs. The kobsvalues for [14C]?G1,6bisP (∼400 s-1)
and [14C]G6P formation are, in contrast, calculated as an
average of the rate of the initial turnover and the rate of the
subsequent equilibration reactions.
?G1,6bisP Is an Intermediate in the ?PGM-Catalyzed
Reaction. In the previous section, we reported that the
reaction between ?G1,6bisP and ?PGM generates the active
form of the enzyme (E-P) and a mixture of ?G1P and G6P.
To demonstrate the intermediacy of ?G1,6bisP in the ?PGM-
catalyzed conversion of ?G1P to G6P, the accumulation of
[14C]?G1,6bisP during a single turnover of the E-P([14C]?G1P)
complex was measured. The first reaction was carried out
with excess ?PGM (40 µM) and limiting ?G1,6bisP (5 µM)
and [14C]?G1P (5 µM). The reaction sequence is shown in
Figure 5 along with the time courses for [14C]?G1P
consumption and the formation of [14C]?G1,6bisP and [14C]-
G6P. The time course of Figure 5B shows that the E‚
[14C]?G1,6bisP complex accumulates to a maximum of 12%
at ∼0.1 s and then declines over the next 0.1 s to an
FIGURE 4: (A) The reaction scheme for the single turnover reaction of ?PGM and [14C(U)]?G1,6bisP. (B) The time course for the single
turnover reaction of ?PGM (40 µM) and [14C(U)]?G1,6bisP (5 µM) in 50 mM K+Hepes containing 2 mM MgCl2(pH 7.0, 25 °C). kobs)
400 ( 20 s-1for ?G1,6bisP consumption; kobs) 600 ( 100 s-1for G1P formation; kobs) 340 ( 40 s-1for G6P formation. (C) The time
course for the single turnover reaction of ?PGM (20 µM) and [14C(U)] ?G1,6bisP (10 µM) in 50 mM K+Hepes containing 2 mM MgCl2
(pH 7.0, 25 °C). kobs) 280 ( 20 s-1for ?G1,6bisP consumption; kobs) 800 ( 500 s-1for G1P formation; kobs) 190 ( 20 s-1for G6P
formation. [14C]?G1,6bisP, (0); [14C]?G6P, ((); [14C]?G1P, (b). The data were fitted to first-order rate eqs 2 and 3.
?-Phosphoglucomutase Catalysis
Biochemistry, Vol. 45, No. 25, 2006 7821

Page 5

equilibrium level of 3%. These results indicate that the
[14C]?G1P is first converted to [14C]?G1,6bisP which in turn
is converted to [14C]G6P.
?G1,6bisP Reorientation. For the ?PGM to complete a
full catalytic cycle, the ?G1,6bisP formed from the reaction
of ?G1P and E-P must become reoriented in the active site
of the free enzyme so that the C(1)phosphate group can
phosphorylate the Asp8. The question addressed in this
section is whether the ?G1,6bisP “flips” while still contained
within the active site or the ?G1,6bisP dissociates into solvent
and then binds to the vacated active site in the opposite
orientation. To test the mechanism of reorientation, a single
turnover of [14C]?G1P catalyzed by excess E-P was carried
out in the presence of unlabeled ?G1,6bisP. If there is no
exchange of the [14C]?G1,6bisP with the unlabeled ?G1,-
6bisP, then the radioactivity will be localized in the G6P
fraction and none will appear in the G1,6bisP fraction. If,
for every turnover, the [14C]?G1,6bisP must leave the
enzyme and mix with the unlabeled ?G1,6bisP in the solvent,
the14C-label will become distributed between the ?G1,6bisP
and the G6P. Specifically, the ratio of14C-label in the G6P
and ?G1,6bisP fractions will be equal to the molar ratio of
the [14C]?G1P and ?G1,6bisP used in the reaction.3
The distribution of radiolabel from [14C]?G1P to ?G1,-
6bisP and the G6P was measured using three different
reaction conditions (see Figure 6). First, 10 µM ?PGM; 20
µM unlabeled ?G1,6bisP; and 10 µM [14C]?G1P were
reacted. The amount of radiolabel in the ?G1,6bisP versus
G6P fractions should be 1:1, consistent with the 1:1 molar
ratio of ?G1,6bisP and ?G1P used in the reaction. A 1:1
ratio of radiolabel is evidenced by the time course shown in
Figure 6A. Second, 10 µM ?PGM; 40 µM unlabeled ?G1,-
6bisP; and 10 µM [14C]?G1P were reacted. The amount of
3This relationship accounts for the fact that the E-P is formed from
free enzyme plus the unlabeled ?G1,6bisP added, and hence, the amount
of ?G1,6bisP in the solution is equal to the ?G1,6bisP added minus
the enzyme added.
FIGURE 5: Scheme for the activation and catalyzed reaction of ?PGM and time course for the single turnover reaction of 40 µM ?PGM
with 5 µM ?G1,6bisP and 5 µM [14C]?G1P in 50 mM K+Hepes (pH 7.0, 25 °C) containing 2 mM MgCl2. (A) Time course for [14C]?G1P
(b) consumption and [14C]G6P (() formation. (B) Time course for [14C]?G1,6bisP formation and consumption.
FIGURE 6: Time course of single turnover reactions of (A) 10 µM ?PGM, 10 µM [14C]?G1P, and 20 µM ?G1,6bisP; (B) 10 µM ?PGM,
10 µM [14C]?G1P, and 40 µM ?G1,6bisP; and (C) 40 µM ?PGM, 5 µM [14C]?G1P, and 50 µM ?G1,6bisP in solutions containing and 2
mM MgCl2in 50 mM K+Hepes (pH 7.0, 25 °C). [14C]?G1,6bisP, (0); [14C]?G6P, ((); [14C]?G1P, (b). The rate data were fitted to first-
order rate eqs 2 and 3.
7822 Biochemistry, Vol. 45, No. 25, 2006
Dai et al.