Báo cáo Y học: Recombinant human glucose-6-phosphate dehydrogenase Evidence for a rapid-equilibrium random-order mechanism

doi:10.1046/j.1432-1033.2002.03015.x

Eur. J. Biochem. 269, 3417–3424 (2002) (cid:1) FEBS 2002

Recombinant 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,* 1Department 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

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 rapid-equilibrium random-order mechanism; kinetics; alternative substrate; product inhibition.

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 lM calculated 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

(EC 1.l.1.49)

dehydrogenase

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.

in Glucose-6-phosphate humans is an X-chromosome-linked housekeeping enzyme, vital for the life of every cell. It catalyses the oxidation of D-glucose 6-phosphate to D-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 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 check of 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)

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dehydrogenase gene was cloned and over-expressed in Escherichia coli so 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

100 lgÆmL)1 ampicillin 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 D600 of 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 (0.1 M Tris/HCl, 5 mM EDTA, 3 mM MgCl2, pH 7.6. with 1 mM e-amino-n-caproic acid, 0.5 mM PMSF, 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 lM NADP+ in this same buffer and assessed according to the WHO guidelines [22] (data not shown). Purity was verified by 10% SDS/PAGE [23].

M

M

M

)1Æcm)1).

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-phosphate (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 (e260 ¼ )1Æcm)1), NADPH at 18.0 · 103 340 nm (e340 ¼ )1Æcm)1) and deaminoNADP+ at 249 nm 6.22 · 103 (e249 ¼ 14.7 · 103

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 fluorescence 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].

Construction of the expression plasmid

Measurements of steady-state kinetic parameters

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.

The purified enzyme was dialysed extensively against equilibration buffer to remove the NADP+ used in chromatography. The reaction mixture for activity assays contained 0.01 M MgCl2, 0.1 M 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 25 (cid:4)C 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 (cid:4)C).

Expression and purification of human recombinant Glc6Pdehydrogenase

The initial-rate equation for the two-substrate reaction catalysed by Glc6P dehydrogenase, in the nomenclature of Dalziel [25], is of the form:

þ

þ

ð1Þ

¼ Uo þ

e m

UX ½X(cid:4)

UY ½Y(cid:4)

UXY ½X(cid:4)½Y(cid:4)

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 [0.1 M KH2PO4, 0.015 M (NH4)2SO4, 0.8 mM MgSO4, 2 lM FeSO4 was adjusted to pH 7.0 with KOH, and 4 mgÆmL)1 glucose, 25 lgÆmL)1 methionine and containing as histidine were

supplements]

added

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

Human glucose-6-phosphate dehydrogenase mechanism (Eur. J. Biochem. 269) 3419

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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

reciprocal 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 the SIGMA PLOT package. In theory, for scattered data, a simple linear fit may introduce a false weighting. Here, however, an internal check was 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

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].

In product inhibition assays, the initial rates were measured for a series of NADPH concentrations (0–20 lM) with 60 lM Glc6P 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 lM and 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 FE and FEL are 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:

ð2Þ

DF ¼ FE (cid:7) F ¼ FEL (cid:7) FE (cid:7)

(cid:8) Kd

DF ½L(cid:4)

The value of Kd can 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] (cid:9) [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

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.

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The secondary plots (Fig. 1B,C), also linear, yielded the shown in kinetic constants and Dalziel parameters Tables 1–3. The initial-rate behaviour gives no indication of any complexities in NADP+ binding.

Alternative substrates

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 Km vs. 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 lM calculated 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 Km for Glc6P obtained here is 50.5 lM, similar to 54.8 lM calculated from the Dalziel parameters in Tables 1–3. The Ki determined for glucosamine 6-phosphate under these conditions was 1.08 mM.

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 /deoxyGlc6P and /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

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 lM for NADP+. As for the kinetic measurements, the

Product regarding enzyme reaction mechanism.

inhibition patterns also offer useful evidence In this case,

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+ (lMÆs) /Glc6P (lMÆs) /NADP+Glc6P 2Æs) (lM kcat (s)1) /NADP+Glc6P/ /NADP+ (lM) /NADP+Glc6P/ /Glc6P (lM) /NADP+Glc6P/ /Glc6P/NADP+ (s)1)

0.0062 ± 0.0004 0.0062 ± 0.0002 0.0062 0.042 ± 0.004 0.042 ± 0.0005 0.042 0.34 ± 0.007 2.3 ± 0.07 2.3 ± 0.03 0.34 ± 0.01 2.3 0.34 54.8 54.8 54.8 6.76 6.76 6.76 161 161 161 161 161 161 1 2 3

Table 2. Dalziel parameters and their ratios for the reaction for substrates: deaminoNADP+ and Glc6P. See legend to Table 1.

/deamino-

NADP+Glc6P 2Æs) (lM

Row no. /o (s) /deaminoNADP+ (lMÆs) /Glc6P (lMÆs) kcat (s)1) /deaminoNADP+Glc6P/ /deaminoNADP+ (lM) /deamino- NADP+Glc6P/ /Glc6P (lM) /deaminoNADP+Glc6P/ /Glc6P/deaminoNADP+ (s)1)

0.014 ± 0.003 0.13 ± 0.016 0.014 ± 0.007 0.13 ± 0.005 0.014 0.13 1.6 ± 0.06 1.6 ± 0.03 1.6 8.74 ± 0.29 67 67 8.72 ± 0.2 67 8.73 5.5 5.5 5.5 42 42 42 71.4 71.4 71.4 1 2 3

Table 3. Dalziel parameters and their ratios for the reaction for substrates: NADP+ and deoxyGlc6P. See legend to Table 1.

/o (s) /NADP+ (lMÆs) /deoxyGlc6P (lMÆs) kcat (s)1) Row no. /NADP+deoxyGlc6P/ /NADP+ (lM) /NADP+deoxyGlc6P/ /deoxyGlc6P (lM) /NADP+deoxyGlc6P/ /deoxyGlc6P/NADP+ (s)1) /NADP+deoxy 2Æs) Glc6P (lM

0.031 ± 0.003 0.27 ± 0.01 68 ± 2.5 0.031 ± 0.001 0.28 ± 0.005 68 ± 1.9 0.031 0.28 68 452 ± 14 451 ± 8.2 451 1674 1611 1643 6.65 6.63 6.64 25 24 25 32.3 32.3 32.3 1 2 3

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titration appears to reflect the behaviour of only one type of NADP+ binding site. In contrast to the effect of coenzyme, Glc6P produced negligible quenching.

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].

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].

to the enzyme, /XY//Y gives Kd,

For a compulsory-order mechanism with substrate X the substrate X leaving the

binding first dissociation constant

for

binary-enzyme complex EX, and this value should not change if an alternative substrate Y is used (Table 2). Similarly, the values of /X and /XY//X/Y should remain unchanged regardless of the nature of substrate Y, even though there may be substantial changes to the individual values of /o, /Y and /XY [25,29]. There is a correspond- ing set of relationships if Y is the leading 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, /XY//X/Y also reveals very different

Fig. 3. Product inhibition of the Glc6P dehydrogenase reaction by NADPH: varied [NADP+]. (A) Lineweaver–Burk plots at a fixed concentration of 60 lM Glc6P and varying concentrations of NADP+ in the presence of a range of NADPH concentrations as indicated. (B) Secondary replot of Kmapp vs. the concentrations of NADPH.

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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:

(cid:1)

(cid:2)

1 þ

¼

þ

þ

ð3Þ

1 k

e m

KYðXÞ ½Y(cid:4)

KXKYðXÞ ½X(cid:4)½Y(cid:4)

KYðXÞKX KY½X(cid:4)

This predicts linear Lineweaver–Burk plots 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 function [31] of the concentration of substrate X:

ð4Þ

½B(cid:4) ¼

b0 þ b1½X(cid:4) þ b2½X(cid:4)2 a2½X(cid:4)2 þ a1½X(cid:4)

Fig. 4. Product inhibition of the Glc6P dehydrogenase reaction: varied [Glc6P]. Lineweaver–Burk plots at a fixed concentration of 10 lM NADP+ and varying concentrations of Glc6P in the presence of a range of NADPH concentrations as indicated. Fig. 6. Determination of dissociation constant of NADP+ by fluores- cence titration. DF ¼ F(E) ) F.

values for the two sugar phosphate substrates (161 s)1 vs. 25 s)1). A compulsory-order mechanism with NADP+ as the leading substrate would therefore appear to be ruled out.

Owing to the higher-order dependence on substrate concentration, the Lineweaver–Burk plot 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. 5. Dead-end inhibition of the Glc6P dehydrogenase reaction by glucosamine 6-phosphate. Lineweaver–Burk plots at a fixed concen- tration of 60 lM Glc6P and varying concentrations of NADP+ in the presence of a range of glucosamine 6-phosphate concentrations as indicated.

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Table 4. Invariant / coefficients predicted for alternative A¢ and B¢.

Alternative substrate

Mechanism A¢ B¢ 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. None /A, /AB//B

R E F E R E N C E S

ture implicit in the steady-state mechanism may be difficult to detect.

Compulsory ordered (A as leading substrate) Theorell–Chance Rapid equilibrium random Ping Pong None /AB//A /B /o, /A, /AB//B /AB//B /A 1. Gaetani, G.F., Galiano, S., Canepa, L., Ferraris, A.M. & Kirk- man, H.N. (1989) Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human ery- throcytes. Blood 73, 334–339.

2. Salvemini, F., Franze, A., Iervolino, A., Filosa, A.M., Salenzano, S. & Ursini, M.V. (1999) Enhanced glutathione levels and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression. J. Biol. Chem. 274, 2750–2757.

3. Kirkman, H.N. & Gaetani, G.F. (1984) Catalase: a tetrameric enzyme with four tightly bound molecules of NADPH. Proc. Natl Acad. Sci. USA 81, 4343–4347.

4. Vulliamy, T., Luzzatto, L., Hirono, A. & Beutler, E. (1997) glucose-6-phosphate important mutations: Hematologically dehydrogenase. Blood Cells Mols. Dis 23, 302–313. 5. Kirkman, H.N., Galiano, S. & Gaetani, G.F. (1987) The function of catalase-bound NADPH. J. Biol. Chem. 262, 660–666.

6. Rowland, P., Basak, A.K., Gover, S., Levy, H.R. & Adams, M.J. (1994) The three dimensional structure of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides refined at 2A˚ resolution. Structure 2, 1073–1087.

As can be deduced from Eqn (3), in a rapid-equilibrium random-order mechanism, the values /XY//Y and /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//Glc6P is 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 lM obtained for /deaminoNADP+Glc6P//deaminoNADP+. Thus the predictions for both substrates are adequately met for this mechanism.

7. Olive, C., Geroch, M.E. & Levy, H.R. (1971) Glucose 6-phos- phate dehydrogenase from Leuconostoc mesenteroides. Kinetic studies. J. Biol. Chem. 246, 2047–2057.

8. Levy, H.R., Ingulli, J. & Afolayan, A. (1977) Identification of essential arginine residues in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. J. Biol. Chem. 252, 3745–3751. 9. Haghighi, B., Flynn, T.G. & Levy, H.R. (1982) Glucose 6-phos- phate dehydrogenase from Leuconostoc mesenteroides. Isolation and sequence of a peptide containing an essential lysine. Bio- chemistry 21, 6415–6420.

indicating that

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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) 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.

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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.

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

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