Engel3,* 1 Department of Biochemistry, The University of Hong Kong, Hong Kong SAR;2Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratory, London, UK;3Dep
Trang 1Recombinant human glucose-6-phosphate dehydrogenase
Evidence for a rapid-equilibrium random-order mechanism
Xiao-Tao Wang1, Shannon W N Au1,2, Veronica M S Lam1,* and Paul C Engel3,*
1
Department of Biochemistry, The University of Hong Kong, Hong Kong SAR;2Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratory, London, UK;3Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland
Cloning and over-expression of human glucose 6-phosphate
dehydrogenase (Glc6P dehydrogenase) has for the first time
allowed a detailed kinetic study of a preparation that is
genetically homogeneous and in which all the protein
mol-ecules are of identical age The steady-state kinetics of the
recombinant enzyme, studied by fluorimetric initial-rate
measurements, gave converging linear Lineweaver–Burk
plots as expected for a ternary-complex mechanism Patterns
of product and dead-end inhibition indicated that the
enzyme can bind NADP+and Glc6P separately to form
binary complexes, suggesting a random-order mechanism
The Kd value for the binding of NADP+ measured by
titration of protein fluorescence is 8.0 lM, close to the value
of 6.8 lMcalculated from the kinetic data on the assumption
of a rapid-equilibrium random-order mechanism Strong
evidence for this mechanism and against either of the
com-pulsory-order possibilities is provided by repeating the
kinetic analysis with each of the natural substrates replaced
in turn by structural analogues A full kinetic analysis was carried out with deaminoNADP+and with deoxyglucose 6-phosphate as the alternative substrates In each case the calculated dissociation constant upon switching a substrate
in a random-order mechanism (e.g that for NADP+upon changing the sugar phosphate) was indeed constant within experimental error as expected The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms Our study appears to provide unambiguous evidence for the latter pattern of substrate binding
Keywords: glucose-6-phosphate dehydrogenase; steady-state kinetics; rapid-equilibrium random-order mechanism; alternative substrate; product inhibition
Glucose-6-phosphate dehydrogenase (EC 1.l.1.49) in
humans is an X-chromosome-linked housekeeping enzyme,
vital for the life of every cell It catalyses the oxidation of
D-glucose 6-phosphate toD-glucono-d-lactone 6-phosphate
in the first committed step of the pentose phosphate pathway,
which provides cells with pentoses and reducing power in the
form of NADPH In red blood cells, this is the only source of
NADPH required to protect the cells (via glutathione [1,2]
and catalase [3,4]) against hydrogen peroxide and other
oxidative damage Accordingly, numerous Glc6P
dehydrog-enase mutations are associated with haemolytic anaemia [5]
Until recently, detailed structural information was
avail-able only for the Glc6P dehydrogenase of Leuconostoc
mesenteroides[6] Extensive kinetic analysis of the NAD+
-and NADP+-linked reactions for this bacterial Glc6P
dehydrogenase [7–10] suggests different mechanisms for the
two coenzymes NADPH inhibition of the NADP+-linked
reaction was competitive with respect to NADP+ and
noncompetitive with respect to Glc6P, whereas inhibition of
the NAD+-linked reaction by NADH was noncompetitive with respect to both NAD+and Glc6P [7] These and other results [8–10] were consistent with a steady-state random-mechanism for the NAD+-linked reaction and an ordered, sequential reaction mechanism, with NADP+binding first, for the NADP+-linked reaction On the other hand, early studies of human Glc6P dehydrogenase have left the kinetic mechanism a matter of controversy, both because of the conflicting conclusions of various investigators [11–13] and because of inherent doubts about Glc6P dehydrogenase purified from pooled, expired blood from a genetically heterogeneous population Adediran [14] proposed an ordered-sequential mechanism with NADP+as the leading substrate, whereas Birke et al [15] obtained quite different results from similar experiments Their steady-state kinetic study, including measurements with inhibitors and alter-native substrates, suggested a random-order ternary-com-plex mechanism
The present study was prompted not only by these unresolved disagreements but also by the need for a reliable kinetic description of the normal enzyme as a baseline for future studies of clinically significant Glc6P dehydrogenase mutants Furthermore, our recently solved crystallographic structure of human Glc6P dehydrogenase [16,17] clearly vindicates earlier claims that each Glc6P dehydrogenase subunit has not one but two coenzyme binding sites, and this
in itself demands a careful checkof the dependence of reaction rates on coenzyme concentration For this reinves-tigation of the kinetic mechanism, the human Glc6P
Correspondence to P Engel, Department of Biochemistry,
University College Dublin, Belfield, Dublin 4, Ireland.
Fax: + 353 1283 7211, Tel.: + 353 1716 1547,
E-mail: paul.engel@ucd.ie
Abbreviations: Glc6P, glucose 6-phosphate.
*Note: these authors contributed equally to this paper.
(Received 12 February 2002, revised 10 May 2002,
accepted 23 May 2002)
Trang 2dehydrogenase gene was cloned and over-expressed in
Escherichia coliso that rate measurements could be made
with freshly prepared, kinetically homogeneous enzyme
Studies of the reaction both with and without added
dead-end or product inhibitors were supplemented with
fluores-cence titration studies to show clearly that human Glc6P
dehydrogenase obeys a rapid-equilibrium random-order
mechanism
M A T E R I A L S A N D M E T H O D S
Enzymes and substrates
Restriction enzymes, calf intestinal alkaline phosphatase,
Sequenase version 2.0 DNA sequencing kit, CircumVentTM
thermal cycle dideoxy DNA sequencing kit and other DNA
modifying enzymes for cloning and DNA markers were all
purchased from New England Biolabs
Glucose-6-phos-phate (Glc6P), 2-deoxyglucose-6-phosGlucose-6-phos-phate (deoxyGlc6P),
glucosamine 6-phosphate and deaminoNADP+(more than
95% purity) were obtained from Sigma Chemical
Com-pany Boehringer Mannheim (now Roche Diagnostics)
supplied NADP+(grade II) and NADPH (grade I)
Oxid-ized coenzymes (NADP+ and deaminoNADP+) were
repurified on DE-32 columns [18] NADP+concentrations
were determined spectrophotometrically at 260 nm (e260¼
18.0· 103
M )1Æcm)1), NADPH at 340 nm (e340¼
6.22· 103
M )1Æcm)1) and deaminoNADP+ at 249 nm
(e249¼ 14.7 · 103
M )1Æcm)1)
Construction of the expression plasmid
All DNA manipulations were carried out by standard
procedures [19] In order to construct a plasmid encoding the
entire human Glc6P dehydrogenase, the 5¢ end of the
full-length cDNA clone pGD-T-5B [20] was amplified with the
primers 5¢-GATGTCAGCCACTGTGGG-3¢ and 5¢-GAC
AGCGCCATGGCAGAGCA-3¢ This introduced an NcoI
site including the Glc6P dehydrogenase initiation codon to
facilitate subsequent manipulations Digestion of the
ampli-fied fragment with NcoI and BamHI produced a 42-bp
fragment, and this was cloned into the expression vector,
pTrc99A, which has an inducible Trc promoter
(Pharma-cia) The resultant plasmid was cleaved with BamHI/SalI
Meanwhile, the cDNA clone pGD-T-5B was digested with
BamHI/XhoI, and the 1780-bp fragment, which corresponds
to most of the Glc6P dehydrogenase cDNA, including the 3¢
coding region, was ligated into the BamHI/SalI-cleaved
pTrc99A The recombinant plasmid, designated pTrc/
G6PD, was shown by dideoxy sequencing to contain the
complete human Glc6P dehydrogenase coding sequence
Expression and purification of human recombinant
Glc6P dehydrogenase
The expression constructs were transformed into E coli
strain DF213 [D(eda-zwf)15, hisGl, rpsL115, metA28, mu+],
which is Glc6P dehydrogenase deficient (E coli Stock
Centre, Yale University) Two hundred milliliters MM63
minimal medium [0.1M KH2PO4, 0.015 M (NH4)2SO4,
0.8 mMMgSO4, 2 lMFeSO4was adjusted to pH 7.0 with
KOH, and 4 mgÆmL)1glucose, 25 lgÆmL)1methionine and
histidine were added as supplements] containing
100 lgÆmL)1ampicillin was inoculated with 1 : 100 of an overnight culture of E coli containing the recombinant Glc6P dehydrogenase plasmid, pTrc/G6PD When the culture reached a D600of 0.4–0.5, 4 mM isopropyl
thio-b-D-galactoside was added to induce synthesis of human Glc6P dehydrogenase Harvested bacteria were resuspended
in 10 mL extraction buffer (0.1MTris/HCl, 5 mMEDTA,
3 mM MgCl2, pH 7.6 with 1 mMe-amino-n-caproic acid, 0.5 mMPMSF, 3 lg/mL aprotinin and 0.1% 2-mercapto-ethanol) and broken by sonication The supernatant was loaded onto a 2¢5¢-ADP Sepharose 4B column (1.0 · 20cm) [21] equilibrated with 0.1 M Tris/HCl buffer, pH 7.6 containing 5% glycerol, 1 mM 6-amino-n-caproic acid,
3 lgÆmL)1 aprotinin and 0.1% 2-mercaptoethanol The enzyme was eluted with 80 lMNADP+in this same buffer and assessed according to the WHO guidelines [22] (data not shown) Purity was verified by 10% SDS/PAGE [23]
Calibration of the fluorescence emitted by NADPH and deaminoNADPH
With NADP+or deaminoNADP+as coenzyme, the activity
of Glc6P dehydrogenase was followed via the increasing fluorescence of the reduced coenzyme The measured fluor-escence change has to be related to the fluorfluor-escence of a known concentration of NADPH or deaminoNADPH Because deaminoNADPH of high purity is not available commercially, the kinetic calibration method of Engel & Hornby was used, relying on enzymatic production of known amounts of reduced cofactor in situ [24]
Measurements of steady-state kinetic parameters The purified enzyme was dialysed extensively against equilibration buffer to remove the NADP+ used in chromatography The reaction mixture for activity assays contained 0.01M MgCl2, 0.1M Tris/HCl buffer, pH 8.0, with varying amounts of sugar phosphate and coenzyme in
a total volume of 1 mL The buffer conditions were in accordance with WHO guidelines [22] An appropriate amount of enzyme, typically in 10 lL, was added to initiate reaction Enzyme activity was assayed at 25C with a recording F-4500 spectrofluorimeter (Hitachi) The excita-tion and emission wavelengths were 340 nm and 450 nm, respectively, with 10 nm slit widths for both lightpaths The working power of the lamp was 700 W To ensure unambiguous initial rate measurements, the enzyme addi-tion was adjusted to give a linear fluorescence increase for at least the first 2 min of reaction Duplicates agreed to within 5% or better On the day of each experiment, the specific activity of the enzyme was checked (WHO method) to confirm stability during storage (20% glycerol,)70 C) The initial-rate equation for the two-substrate reaction catalysed by Glc6P dehydrogenase, in the nomenclature of Dalziel [25], is of the form:
e
m¼ UoþUX
½Xþ
UY
½Yþ
UXY
where X and Y are sugar phosphate and coenzyme, respectively The four / parameters are obtained from initial-rate measurements at varying concentrations of X for
a series of fixed concentrations of Y Rearrangement of the equation shows that the intercepts of primary double
Trang 3reciprocal plots with l/[X] as the variable, for example, are
given by /0+ /Y/[Y] and the slopes by /X+ /XY/[Y]
The secondary plots of these slopes and intercepts against
l/[Y] provide estimates for the individual initial-rate
para-meters [25] In the present case, the lines in the primary plots
were drawn by least-squares linear fit using theSIGMA PLOT
package In theory, for scattered data, a simple linear fit
may introduce a false weighting Here, however, an internal
checkwas applied by using both possible plotting sequences
to extract the kinetic parameters, i.e using both 1/[X] and
1/[Y] as alternative variables for the primary plots The close
correspondence in Table 4 and the good linearity observed
throughout argue against any serious error introduced by
the graphical procedures
Inhibition studies
In product inhibition assays, the initial rates were measured
for a series of NADPH concentrations (0–20 lM) with
60 lMGlc6P and NADP+concentrations varied from 2 lM
to 50 lM A similar experiment was carried out by varying
the Glc6P concentrations from 15 lM to 150 lMand the
NADPH concentrations again from 0 lM to 20 lM while
fixing the NADP+concentration at 10 lM In analogous
fashion, glucosamine 6-phosphate was used as an inhibitor,
covering the same combinations and ranges of substrate
concentration as used in the experiments with NADPH
Fluorescence titration studies
Additions of NADP+partially quenched the fluorescence
at 345 nm emitted when purified Glc6P dehydrogenase was
excited at 290 nm If FEand FELare the relative
fluores-cence intensities of enzyme, E, and enzyme-ligand complex,
EL, F is the measured fluorescence at a concentration [L] of
the ligand, and Kd is the dissociation constant of the
complex, it can be shown that:
DF¼ FE F ¼ FEL FEDF
½L Kd ð2Þ The value of Kdcan thus be obtained from the negative
slope of a plot of DF/[L] against DF Provided that total
concentration of L, free and bound, [L]T, is much higher
than the total enzyme concentration ([E]T), it can be taken
that [L] [L]T
R E S U L T S
Enzyme preparation
Chromatography on 2¢5¢-ADP Sepharose 4B yielded
recombinant human Glc6P dehydrogenase of 99% purity
or better as judged by SDS/PAGE (data not shown) The specific activity was about 100 UÆmg)1 protein Typically about 5 mg of purified enzyme could be obtained from 1 L
of E coli culture This enzyme behaved identically to Glc6P dehydrogenase from human cells and showed identical mobility in native gel electrophoresis (data not shown) This agrees with the finding of Bautista et al [26] that recombinant human Glc6P dehydrogenase expressed
in E coli behaves similarly to the authentic enzyme from red cells
Initial velocity experiments The strictly linear and converging double reciprocal plots obtained with different combinations of Glc6P and NADP+(Fig 1) are consistent with a sequential mechan-ism, in which both substrates must bind to the enzyme simultaneously before product formation can occur [25,27]
Fig 1 Graphs to determine the various / parameters for the reaction catalysed by human Glc6P dehydrogenase with Glc6P and NADP + as substrates (A) Primary plots of e/v vs 1/[NADP+] at nine fixed con-centrations of Glc6P (B) Secondary plots of slopes of primary plots vs 1/[Glc6P] (C) Secondary plots of intercepts of primary plots vs 1/ [Glc6P].
Scheme 1.
Trang 4The secondary plots (Fig 1B,C), also linear, yielded the
kinetic constants and Dalziel parameters shown in
Tables 1–3 The initial-rate behaviour gives no indication
of any complexities in NADP+binding
Alternative substrates
An alternative substrate, when available, can be a useful
tool for differentiating kinetic models, as first reported by
Wong & Hanes [28] The alternative substrate and
coen-zyme used here are deoxyGlc6P and deaminoNADP+, and
the corresponding kinetic parameters are given in Tables 2
and 3 Sample data for deoxyGlc6P are shown in Fig 2 A
comparison of the values of 1//o(¼ kcat) shows that the
enzyme is more active under optimal conditions with its
natural substrates The decrease in rate is much more
marked, however, with deoxyglucose 6-phosphate than with
the coenzyme analogue, for which the factor of increase in
individual / constants is at most fourfold to fivefold
(/Glc6P) At lower concentrations of sugar phosphate, the
contrast between the natural substrate, Glc6P, and the
deoxy analogue is greatly accentuated, and this is reflected
in the very high values of /deoxyGlc6Pand /NADP+ deoxyGlc6P,
which are more than 200-fold larger than the corresponding
parameters for Glc6P (Tables 1–3)
Kinetics of inhibition by NADPH and glucosamine
6-phosphate
Product inhibition patterns also offer useful evidence
regarding enzyme reaction mechanism In this case,
6-phosphogluconolactone is labile and cannot be obtained
at high enough purity for kinetic experiments However, it is possible to determine the effects of NADPH on this reaction (Figs 3 and 4) The intersection on the vertical axis in Fig 3A indicates competitive inhibition with respect to NADP+ The linear secondary plot of the apparent Kmvs inhibitor concentration (Fig 3B) gives (y-intercept) an apparent Km value of 7.08 lM for NADP+ with 60 lM
Glc6P in the absence of inhibitor, in good agreement with the value of 6.76 lMcalculated from the Dalziel parameters
in Tables 1–3, and a negative abscissa intercept of 9.0 lM
for Ki of NADPH Similarly, for Glc6P concentrations varied from 15 lM to 150 lM with a fixed NADP+ concentration of 10 lM, NADPH concentrations from
0 lM to 20 lM gave a general noncompetitive (mixed) inhibition pattern with respect to Glc6P (Fig 4)
Glucosamine 6-phosphate, chosen as a dead-end inhib-itor, was found to be competitive with respect to Glc6P (data not shown) but general noncompetitive (mixed) with respect to NADP+(Fig 5) The apparent Kmfor Glc6P obtained here is 50.5 lM, similar to 54.8 lMcalculated from the Dalziel parameters in Tables 1–3 The Kidetermined for glucosamine 6-phosphate under these conditions was 1.08 mM
Measurement of dissociation constant of NADP+ Figure 6 shows that NADP+quenches the intrinsic fluor-escence of Glc6P dehydrogenase The data are consistent with a simple binding process with a dissociation constant of 8.0 lMfor NADP+ As for the kinetic measurements, the
Table 1 Dalziel parameters and their ratios for the reaction for substrates: NADP + and Glc6P Kinetic data were determined by the use of primary plots against both reciprocal of coenzyme concentration (Row 1) and sugar phosphate concentration (Row 2) The mean value obtained from each plot is also indicated (Row 3) (Standard errors were obtained from the regression of the line of best fit through the data points).
Row
no.
/ o
(s)
/ NADP +
(l M Æs)
/ Glc6P
(l M Æs)
/ NADP + Glc6P
(l M2Æs)
/ NADP + Glc6P / / NADP + (l M )
/ NADP + Glc6P / / Glc6P (l M )
/ NADP + Glc6P / / Glc6P / NADP + (s)1)
k cat
(s)1)
Table 2 Dalziel parameters and their ratios for the reaction for substrates: deaminoNADP+and Glc6P See legend to Table 1.
Row
no.
/ o
(s)
/ deaminoNADP +
(l M Æs)
/Glc6P (l M Æs)
/ deamino-NADP + Glc6P
(l M2Æs)
/ deaminoNADP + Glc6P / / deaminoNADP + (l M )
/ deamino-NADP + Glc6P / / Glc6P (l M )
/ deaminoNADP + Glc6P / / Glc6P / deaminoNADP + (s)1)
k cat
(s)1)
Table 3 Dalziel parameters and their ratios for the reaction for substrates: NADP+and deoxyGlc6P See legend to Table 1.
Row
no.
/ o
(s)
/ NADP +
(l M Æs)
/ deoxyGlc6P
(l M Æs)
/ NADP + deoxy Glc6P (l M2Æs)
/ NADP + deoxyGlc6P / / NADP + (l M )
/ NADP + deoxyGlc6P / / deoxyGlc6P (l M )
/ NADP + deoxyGlc6P / / deoxyGlc6P / NADP + (s)1)
k cat
(s)1)
Trang 5titration appears to reflect the behaviour of only one type of
NADP+binding site In contrast to the effect of coenzyme,
Glc6P produced negligible quenching
D I S C U S S I O N
Human Glc6P dehydrogenase has Km values in the
micromolar concentration range for both the sugar
phos-phate substrate and the coenzyme, and therefore their
reliable estimation requires rate measurements with very
low concentrations of each The fluorimetric method
employed in this study allowed precise and reproducible
initial-rate measurements even for these low concentrations,
permitting a full analysis of all the initial-rate parameters
[25] The primary plots were linear over wide ranges of
concentrations for both substrate and coenzyme The
discovery that there are two NADP+binding sites on the
enzyme [17,30,31] had raised the possibility that at low
coenzyme concentrations both sites might contribute to the
observed overall pattern of binding There was no indication
of any such complexities in these experiments The pattern
of converging lines confirms earlier reports that human
Glc6P dehydrogenase follows a sequential mechanism
[14,15]
For a compulsory-order mechanism with substrate X
binding first to the enzyme, /XY//Y gives Kd, the
dissociation constant for substrate X leaving the
binary-enzyme complex EX, and this value should not change if an alternative substrate Y is used (Table 2) Similarly, the values of /Xand /XY//X/Yshould remain unchanged regardless of the nature of substrate Y, even though there may be substantial changes to the individual values of /o, /Yand /XY[25,29] There is a correspond-ing set of relationships if Y is the leadcorrespond-ing substrate These relationships were tested for Glc6P dehydrogenase by using the alternative substrates deaminoNADP+ and deoxyGlc6P (Tables 1–3) Tables 1 and 3 show the results obtained from the use of alternative sugar phosphate substrates with the same coenzyme, NADP+ If NADP+
is the leading substrate, then /NADP+ Glc6P//Glc6P should
be equal to /NADP+ deoxyGlc6P//deoxyGlc6P The mean values obtained, 6.8 lM and 6.6 lM, respectively, were indeed very similar However, /NADP+ with Glc6P as the substrate is 0.042 lMÆs, which is 6.6-fold lower than /NADP + with deoxyGlc6P as the substrate (0.28 lMÆs) Consequently, / // / also reveals very different
Fig 2 Graphs to determine the various / parameters for the reaction
catalysed by human Glc6P dehydrogenase with deoxyGlc6P and
NADP+as substrates (A) Primary plots of e/v vs 1/[NADP+] at eight
fixed concentrations of deoxyGlc6P (B) Secondary plots of slopes of
primary plots vs 1/[deoxyGlc6P] (C) Secondary plots of intercepts of
primary plots vs 1/[deoxyGlc6P].
Fig 3 Product inhibition of the Glc6P dehydrogenase reaction by NADPH: varied [NADP+] (A) Lineweaver–Burkplots at a fixed concentration of 60 l M Glc6P and varying concentrations of NADP +
in the presence of a range of NADPH concentrations as indicated (B) Secondary replot of K mapp vs the concentrations of NADPH.
Trang 6values for the two sugar phosphate substrates (161 s)1vs.
25 s)1) A compulsory-order mechanism with NADP+as
the leading substrate would therefore appear to be ruled
out
Tests for a mechanism with the sugar phosphate as the leading substrate can similarly be made by comparing the data for alternative coenzymes (Tables 1 and 2) The mean value of /NADP+ Glc6P//NADP+ is 55 lM for NADP+ as coenzyme, which is reasonably close to the value of 67 lM
obtained with deaminoNADP+as the coenzyme However, the mean value of /Glc6P with NADP+as the coenzyme (0.34 lMÆs) is about five times lower than /Glc6P with deaminoNADP+as the coenzyme (1.6 lMÆs) Correspond-ingly, the value of /NADP+ Glc6P//NADP+/Glc6P(161 s)1) is quite different from the value of /deaminoNADP + Glc6P/ /deaminoNADP +/Glc6P(42 s)1) It thus seems that a compul-sory-order mechanism with Glc6P as leading substrate is also very unlikely, leaving a rapid-equilibrium random-order sequential mechanism (Scheme 1) as the remaining option
In the reciprocal form of the rate equation:
e
m¼1
k 1þKYðXÞKX
KY½X þ
KYðXÞ
½Y þ
KXKYðXÞ
½X½Y
ð3Þ This predicts linear Lineweaver–Burkplots when the concentration of one substrate is fixed while the other is varied In the more general steady-state random-order mechanism, the reciprocal of the rate at a fixed concentra-tion of substrate Y is a complex funcconcentra-tion [31] of the concentration of substrate X:
½B ¼b0þ b1½X þ b2½X
2
a2½X2þ a1½X ð4Þ Owing to the higher-order dependence on substrate concentration, the Lineweaver–Burkplot would not be strictly linear Because linear plots were observed in all the experiments described here without any indication of a systematic departure from this pattern, this in itself suggests that the reaction catalysed by human Glc6P dehydrogenase may involve a rapid-equilibrium rather than a steady-state random-order mechanism, although admittedly the
curva-Fig 6 Determination of dissociation constant of NADP+by fluores-cence titration DF ¼ F(E) ) F.
Fig 5 Dead-end inhibition of the Glc6P dehydrogenase reaction by
glucosamine 6-phosphate Lineweaver–Burkplots at a fixed
concen-tration of 60 l M Glc6P and varying concentrations of NADP+in the
presence of a range of glucosamine 6-phosphate concentrations as
indicated.
Fig 4 Product inhibition of the Glc6P dehydrogenase reaction: varied
[Glc6P] Lineweaver–Burkplots at a fixed concentration of 10 l M
NADP + and varying concentrations of Glc6P in the presence of a
range of NADPH concentrations as indicated.
Trang 7ture implicit in the steady-state mechanism may be difficult
to detect
As can be deduced from Eqn (3), in a rapid-equilibrium
random-order mechanism, the values /XY//Yand /XY//X
are the dissociation constants for substrates X and Y,
respectively [32] If this is indeed the mechanism of human
Glc6P dehydrogenase, then independent estimates of the
dissociation constant for NADP+ using Glc6P or
deo-xyGlc6P as substrates (Tables 1 and 3) should be equal
(Table 4) Similarly, the dissociation constant for Glc6P
(Tables 1 and 2) should also be the same regardless of
the coenzyme As mentioned earlier, the value of
/NADP+ Glc6P//Glc6Pis 6.8 lM, which is almost the same as
/NADP + deoxyGlc6P//deoxyGlc6P (6.6 lM) This figure is also
very similar to the value of 8.0 lM, independently obtained
by fluorescence titration (Fig 6) Also /NADP + Glc6P/
/NADP+is 55 lM, similar to a value of 67 lMobtained for
/deaminoNADP+ Glc6P//deaminoNADP+ Thus the predictions for
both substrates are adequately met for this mechanism
Independent tests of mechanism may be applied by
using inhibitors In the present study, NADPH was found
to be competitive with respect to NADP+ (Fig 3) and
general noncompetitive with respect to Glc6P (Fig 4)
The dead-end inhibitor, glucosamine 6-phosphate is
competitive with respect to Glc6P and general
noncom-petitive with NADP+ (Fig 5) indicating that this
inhibitor can bind to both the free enzyme and the
enzyme–NADP+ binary complex Because glucosamine
6-phosphate is an analogue of Glc6P, it seems likely that
Glc6P also can bind to both free enzyme and
enzyme-NADP+complex The inhibition studies therefore suggest
that both substrate and coenzyme can bind to the free
enzyme This in itself points towards a random-order
mechanism, further substantiating the quantitative
analy-sis above
In summary, this study offers the first clear
documenta-tion of a rapid-equilibrium random-order mechanism for
normal human Glc6P dehydrogenase The direct
demon-stration of crystal complexes of Glc6P dehydrogenase–
Glc6P and Glc6P dehydrogenase–NADP+ also tends to
support this conclusion (S W N Au, S Gover & M J
Adams, unpublished data) The discrepancy between the
mechanisms deduced in this present study and in some
previous reports could be due to the heterogeneous origin of
the Glc6P dehydrogenase used in earlier work
A C K N O W L E D G E M E N T S
Shannon W N Au was supported by a University of Hong Kong
Postgraduate Studentship and the project was initiated by a seed grant
from the University of Hong Kong Committee on Research Grants.
Xiao-Tao Wang was supported, as a research assistant, by the Faculty
of Medicine Faculty Research Fund, followed by the Hong Kong Research Grant Council HKU 7272/98M Support from the University
of Hong Kong Committee on Research and Conference Grants is also gratefully acknowledged.
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Table 4 Invariant / coefficients predicted for alternative A¢ and B¢.
Mechanism
Alternative substrate
Compulsory ordered None / A , / AB // B
(A as leading substrate)
Theorell–Chance None / o , / A , / AB // B
Rapid equilibrium random / AB // A / AB // B
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