Biochimica et Biophysica Acta, 1122(1992) 99-106

99

© 1992 ElsevierScience Publishers B.V. All rights reserved 0167-4838/92/$05.00

BBAPRO 34240

Unfolding and trypsin inactivation studies reveal a conformation drift of glucose-6-phosphate dehydrogenase upon binding of N.M)P Jos6 M. Bautista

a, Jos6

M. Fuentes b, Amalia Diez and Germ:in Soler b

a,

Carlos Guti6rrez-Merino c

a Departamento de Bioqufmica y Biologfa Molecular IV, Unicersidad Complutense de Madrid, Facultad de Veterinaria, Madrid (Spain), t, Departamenlo de Bioquimica y Biolog[a Molecular, Facultad de Veterinaria, UnirersMad de Extremadura, Cdceres (Spaht) and '" Departamento de Bioqufmica y Bioiogfa Molecular, Facultad de Ciencias, Unicersidad de Extremadura. Badajoz (Spain)

(Received 17 June 1990) (Revised manuscriptreceived311September 1991)

Keywords: Glucose-6-phosphatedehydrogenase;Unfolding;Guanidine hydrochloride;Thermal denaturation;Trypsin;NADP Binding of NADP to glucose-6-phosphate dehydrogenase (G6PD) from Dicentrarchus labrax liver has stabilized its native structure against thermal inactivation, guanidine hydrocbloride unfolding and inactivation by tryptic digestion. The time-course of G6PD inactivation by guanidine hydrochloride in the presence of NADP has provided experimental evidence in favor of a conformational drift upon NADP binding to the bass enzyme. Based on the inactivation patterns obtained when the enzyme was treated with guanidine hydrochloride and trypsin, it is proposed that the enzyme conformation induced upon NADP binding is in slow equilibrium with the conformation stabilized in the absence of NADP. FPLC studies have shown that micromolar concentrations of NADP induced oligomerization of G6PD. In addition, the different K~.5 values of NADP binding to the enzyme, ranging from 1-2 /~M (from trcpsin inactivation) to 90 #M (from titration of the intrinsic fluorescence), suggest a step-wise binding of NADP to the oligo :~ r, with negative cooperativity in the saturation process.

Introduction

Conformational changes have been recognized as a significant feature in enzyme action for a long time and often their main effectors are substrates and coenzymes. Enzyme-substrate and enzyme-coenzyme complexes have been shown in many cases to be more stable against proteolysis and heat [1,2], and a correlation between heat stability and susceptibility to proteolytic action has been found in several proteins [3,4]. The treatment of enzymes with chemical, biochemical or physical agents can result in controlled modification of enzymic activity, either by changes directly affecting the catalytic centre, or via conformational modulations at the active site as a result of chemical alterations occurring elsewhere in the molecule.

Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; GdnHCI, guanidinehydrochloride. Correspondence: J.M. Bautista, Departamentode Bioqu[micay Biological MolecularIV, UniversidadComplutensede Madrid, Facultad de Velerinaria, 28040 Madrid, Spain.

Guanidine hydrochloride as a chaotropic solute is a powerful denaturing agent for proteins. The precise nature of the interactions of guanidine with protein groups is not well understood, but it has been reported that guanidine acts on the protein surface of a model system, liver alcohol dehydrogenase, in addition to being able to diffuse inside the protein matrix [5,6]. The diffusion of guanidine at sub-denaturing concentrations causes a loosening of intramolecular interactions as the secondary structure is being disrupted. Bass G6PD has been reported to display an isomerization process during the catalytic cycle, and its enzymatic activity is regulated by NADPH in a parabolic way, i.e., it is dependent upon the second power of NADPH concentration, being highly sensitive to the N A D P H / N A D P concentration ratio [7]. These steady-state kinetics studies have led us to suggest that significant structural transitions are taking place in the enzyme under the influence of the coenzyme [7]. Moreover, in the most studied G6PD, the human G6PD, there is evidence that NADP modulates the average distribution of different protein conformations [8,9], and that NADP is required for the enzyme stability [10], this effect being concentration dependent [11].

100 Allosteric behaviour of human G6PD has been demonstrated under certain experimental conditions that seem to stabilize the enzyme dimer [12-14]. These considerations have led us to investigate further the effects of NADP binding on the conformational properties of G6PD. This paper reports the treatment of native G6PD from bass liver with several denaturing agents, such as guanidine hydrochloride, temperature, and trypsin, in order to obtain information about the structure flexibility of the protein. In addition, the effect of NADP upon the aggregation state of G6PD has been investigated.

Materials and Methods Glucose 6-phosphate dehydrogenase (G6PD) was purified to homogeneity from bass Dicentrarchus labrax liver as previously described [7]. The assays of G6PD activity were performed at 30°C by following absorbance changes at 340 nm in a Hitachi 150-20 spectrophotometer, using quartz cuvettes with 1.0 cm light path. Unless stated otherwise, the assay mixture contained 0.1 M Tris-HCl (pH 7.6) 0.5 mM NADP and 3 mM glucose 6-phosphate. The linearity of product generation during the time interval used to estimate initial rate values was carefully assessed, i.e., the rates were determined before 10% of the substrate was consumed. The protein concentration was determined following the method of Bradford [15] as modified by Marshall and Williams [16]. G6PD unfolding by Gdn-HCI was studied at 4°C in a incubation mixture containing 0.1 M Tris-HCl (pH 7.6), 1 mM DTI" and 0.2 mM EDTA. Native G6PD (final concentration 0.2/zg/ml) was added at zero time to the incubation mixture containing Gdn-HCI (final concentration varying from 0 to 0.6 M). Aliquots from each incubation mixture were removed a t different times and G6PD activity was assayed. Reproducibility of this experiment was checked by repeating the same twice protocol with a different preparation of enzyme since we were able to carry out no more than two assays at each indicat~-.d time. The given values are the average of percentage enzyme activi~,. G6PD cleavage by trypsin in the presence of NADP was carried out at 4°C in 0.08 M Tris-HCl (pH 7.6), 3.6% glycerol, 2.8 mM /3-mercaptoethanol and 0.16 mM EDTA. The trypsin/dehydrogenase ratio used was 1 : 20 (w/w). From each incubation mixture aliquots were taken at different times, and the proteolytic activity was stopped by adding trypsin inhibitor at a concentration 20-fold greater than the trypsin concentration present in the incubation mixture. The percentage of enzyme inactivation was obtained by duplicate assaying G6PD activity. We carefully assessed the following points in control experiments: (1) trypsin activity was

completely inhibited by the indicated concentration of trypsin inhibitor; (2) the activity of trypsin was constant in these experimental conditions during the time of the handlings; (3) NADP has no effect on trypsin activity; and (4) G6PD is stable in the absence of trypsin during the time course of these experiments. Assays of trypsin activity were run at 30°C following the method of Geiger and Fritz [17], with the reaction mixture 0.1 M Tris-HC! (pH 7.6), 0.35 m g / m l benzoyl-arginine-pnitroanilide and 0.066/~g/ml trypsin. Denaturation of G6PD by heat was carried out in 20 mM Tris-HCl (pH 7.6) 9% glycerol, 7 mM fl-mercaptoethanol and 0.4 mM EDTA, in the presence of glucose 6-phosphate or NADP. The preincubation temperature was varied between 20 and 70°C, and incubation times were 5 or 10 min. After each preincubation, the sampies were placed on ice and the G6PD activity was measured in duplicate. All the experiments were done with enzyme preparations dialysed against the corresponding appropiate buffer. Fluorescence measurements have been made using a Hitachi-Perkin Elmer spectrofluorimeter, mod. 65040, operated in ratio mode. The analysis of fluorescence data has been carried out as indicated in Ref. 18. Briefly, for an equilibrium, E+B~

EB,

Kd=

[E][B] [EB]

(I)

where E is the fluorescent molecule (in this case G6PD) and B is the molecule able to bind to E (in this case NADP or glucose 6-phosphate), it can be written: IE]

lEa]

F = F(E)Z-~-~ + F(EB) gT ET

(2)

with F(E) and F(EB) being the relative fluorescence intensities of E and EB forms, and E T is the total concentration of E. From Eqns. 1 and 2 F ( E ) - F(EB) F- F(EB)

[B] = 1+-Kd

(3)

Eqn. 3 allows to obtain the value of K d from the slope of the linear regression plot of ( F ( E ) - F(EB))/(FF(EB)) versus free B concentration, provided that total B concentration, BT, is much higher than the total enzyme concentration, it can be taken [B] ~ B T. G6PD chromatography was performed on a Superrose 12HR 10/30 column in a Pharmacia fast-protein liquid chromatography (FPLC) system. The buffer for equilibration and elution was Tris-HCl 0.02 M (pH 7.6), 9% glycerol, 7 mM /3-mercaptoethanol and 0.4 mM EDTA. A flow rate of 0.5 m l / m i n for the loading and elution of G6PD was employed.

101 TABLE I

G6PD thermostability as a fimction of the presence of NADP or glucose 6-phosphate (G6P) The incubation time at :he indicated temperature was 5 minutes. Values are expressed as percentage of enzyme activity. Temperature Maximum activity (100%) corresponds to a value of specific activity of 210 I U / m g . E

Incubation condition

Temperature (°C) 20

30

40

50

60

70

Free G6PD G6PD + NADP 0.9 mM G 6 P D + G 6 P 1.36 mM

100 100 I00

100 100 100

88 100 88

44 92 62

2 33 5

0 0 0

N f~ U4

I

Unless stated otherwise, reproducibility of each experiment shown here was checked by repeating the same protocol with a different enzyme preparation. NADP, glucose 6-phosphate and ATP were obtained from Boehringer-Mannheim. Trypsin, benzoylarginine-p-nitroanilide, trypsin inhibitor tyFe II-S from Sigma. Gdn-HCl from Pierce. All other chemicals used were of the highest purity available. Glassobidistilled water was used to prepare all the solutions used in this study. Results

Thermal inactivation of G6PD Saturating concentrations of NADP largely enhanced the thermal stability of purified bass G6PD (Table I). This effect was clearly specific for NADP. Saturating concentrations of glucose 6-phosphate (as much as 1 mM) failed to produce any significant effect on the rate of thermal inactivation of G6PD. In addition, the results shown in Table I indicated a large shift

tt0~

I

~

0.4

I

0.6

Gdn-HCI Concentration Fig. 2. Loss of enzyme activity as a function of Gdn-HCI concentration. Native bass G6PD (0.2/.Lg/ml) was incubated in 0.1 M Tris-HCl (pH 7.6) (containing 1 mM DTT and 0.2 mM EDTA) at 4°C with different levels of Gdn-HCI. After 10 rain of incubation G6PD activity was assayed. Constant rates of enzyme activity were obtained during the 6 rain of G6PD assay.

of the denaturation temperature upon binding of NADP, approx. 10°C. Therefore, we made use of this effect of NADP to obtain the apparent association constant of this coenzyme to the enzyme. At 50°C, (Fig. 1) the G6PD inactivation rate dependance with the NADP concentration gave a Ko.5 of protection against inactivation between 1 and 10 /.tM (as derived by applying the equation of the hyperbolic curve [ K = (ayXb + x)], fitting the obtained curves at 5 and 10 min incubation, respectively). Moreover, these results suggested that the conformation of G6PD was largely altered upon binding of NADP, and that recyc!ing back to the predominant conformation in the absence of NADP was a slow kinetic process. These points were further investigated.

Guanidine hydrochloride inactivation

i

75"

z "

~

J

IO0

=[ >N

0.2

501 E5

N

0 0

I 200

[NADP]

I 400

600

~M

Fig. l. Dependence of G6PD thermostability at 50°C on the concentration of NADP. The different symbols correspond to (e) 5 rain and ( I ) 10 min incubation at 50°C prior to measuring the enzymatic activity. Maximum activity corresponds to a value of specific activity of 190 I U / m g . The concentration of G6PD ranged between 1.8 and 2.0/zg/ml.

As illustrated in Fig. 2, the response of G6PD to Gdn-HCl concentration was monophasic, related to an exponential decay in enzyme activity as Gdn-HC! concentration was increased. In the absence of Gdn-HC! and in these experimental conditions no significant loss of enzyme activity was detected. The inactivation by Gdn-HCI was not reverted upon 12-h dialysis against buffer. Thus, the loss of enzyme activity was associated with denaturation of G6PD, which was found to be irreversible (results not shown). The sensitivity of the G6PD activity to denaturation by Gdn-HCl was clearly higher than that reported for many globular proteins [19], likely reflecting an early step in the disorganization of particular protein domains involved in catalysis.

102 TABLE 11 Kinetic parameter for tile unfolding o f G6PD by 0.5 M Gdn,HCI at 4°C

The values of k r were calculated between 0 and 20 rain and the values of k s between 20 and 60 min. Protein concentration was 0.2 /xg/ml. The enzyme activity values correspond to the residual activity at the end of each phase.

0

20

40

60

80

lO0

120

Time (min)

Fig. 3. Semilog plot of the time course of unfolding as a function of NADP concentration. Native bass G6PD was incubated in the conditions described in the legend to Fig. 2 at a fixed Gdn-HCi concentration of 0.5 M in the presence of the following NADP concentrations: (e) 0/zM; (0) I0/zM; ( • ) 25/aM; (D) I00 #M: (&) 250 p.M; (zx) 500 ttM. At the indicated time aliquots were taken, and G6PD activity was assayed. Constant rates of enzyme activity were obtained during the 6 min of G6PD assay.

[NADP]

I st phase

(/LM)

k[

amplitude (% activity)

ks

2nd phase amplitude (% activity)

0 10 25 100 250

0.264 0.260 0.192 0.119 0.082

91 85 82 62 42

0.040 0.040 0.040 0.023 0.017

9 15 18 38 58

different rate constants. The results of parametric analysis of the data from Fig. 3, obtained by deconvolution of both processes are presented in Table I1, where kf and k~ are the fast and slow inactivation rate constant, respectively. The amplitude of the rapid kinetic process decreased as the NADP concentration was increased and, therefore, the molar fraction of enzyme sensitive to rapid Gdn-HCI inactivation decreased as the NADP was increased. To study the selectivity of this conformational drift, we also tested the effect of glucose 6-phosphate and ATP. Both failed to show protection against Gdn-HCI inactivation. Thus, the binding of ATP and glucose 6-phosphate to the catalytic centre [7,20] did not produce any significant conformational change.

When different Gdn-HCI concentrations were tested for G6PD inactivation, 0.5 M Gdn-HCI proved to be optimal to follow the time-course of unfolding (Fig. 3). After 5 min incubation in the absence of NADP, the enzyme lost approx. 25% of its initial activity; in the presence of 250 /~M NADP the loss of activity was negligible even after 30 min incubation. The timecourse of inactivation (Fig. 3) fitted to the sum of two independent first-order kinetic processes, both with

4.5

•~

40

~5 ~

:i.5

30 L

i

I

I

J

I

i

i

•

0

1

2

3

4

5

6

7

8

Time

Ihours)

Fig. 4. Semilog plot of the time course of trypsin inactivation as a function of NADP concentration. Cleavage was performed at 4°C. Reaction mixtures included 20/xg of enzyme and 1 #g of trypsin in 0.08 M Tris-HCi (pH 7.6) in a vol. of 750 #l. The NADP concentrations used were: (e) 0 #M; (o) 0,5 tzM; (r~) 1/.tM; ( 1, ) 10 FtM; ( • ) 100/zM. Aliquots were taken at the indicated time and G6PD activity assayed. Constant rates of enzyme activity were obtained during the 6 min of G6PD assay.

103

Inactication by Trypsin digestion

TABLE 1II

Since trypsin finally renders denatured proteins following a distinct pathway from that of Gdn-HCl, we attempted to assess further the hypothesis of a NADP-induced conformational change in G6PD. Incubation of G6PD with trypsin during 30 min in the absence of NADP resulted in the loss of approx. 50% of its initial activity. Micromolar NADP concentrations largely protected the enzyme against trypsin digestion, e.g., in the presence of 0.5 p.M NADP G6PD maintained about 70% of the starting activity after 30 rain of incubation. Fig. 4 illustrates the time-course of G6PD inactivation by trypsin at 4°(2 with a dehydrogenase/proteinase ratio of 1 : 20. The pattern of G6PD inactivation by trypsin obtained in the presence of glucose 6-phosphate and ATP did not show significant differences from the pattern obtained in the control experiment (no additions). These results further strengthened the hypothesis that trypsin cleaves G6PD at a point close to or overlapping with the NADP binding domain in the enzyme and as a result it produced rapid inactivation. The time-course of inactivation followed two independent first-order kinetic processes whose rate constants were obtained as outlined above and are listed in Table III. Up to 20 min there was a first, fast phase of inactivation, in which the semilog plots of activity vs. time had a slope roughly independent of the NADP concentration. In contrast, the amplitude of the slow kinetic phase (after 20 min) was strongly dependent upon NADP concentration in the range 0-100 p.M, and this rate of inactivation was not largely altered by these NADP concentrations. This behaviour was parallel to that observed when G6PD was treated with Gdn-HCl.

Kinetic parameter for the inat'tit,ation of G6PD by trypsin at 4°C

15

• "1

1.

.-" I

O. O b_ Ix.

06 03

0

100

200

300

400

500

[ NADP l, I..IM

Fig. 5. Dependence of the intrinsic fluorescenceof bass G6PD on NADP concentration. The intensityof fluorescence of bass G6PD has been normalizedto the fluorescencein the absence of NADP. Temperature: (o) 25°C and (o) 40°C. Protein concentration:60-70 #g/ml. Inset: Plot of ~t Fr,a~/~F vs. the concentrationof NADP. ~Fmax = Fa - Fmin, and 5F =/7o - F, where Fa, F and Fmin represent the intensityof fluorescencein the absenceof NADP, at a given NADP concentrationand at saturationof NADP, respectively.

The values of k t were calculated between 0 and 20 min and the values of k~ between 20 and 480 min. The given enzyme activity values correspond to the residual activity at the end of each phase. Protein concentration was 27 txg/ml. [NADP]

1st phase

(/zM)

kf

amplitude (% activity)

k~

amplitude (% activity)

0.073 0.025 0.016

62 36 29

38 64 71 100 IO0

0.0 0.5 1.5 10.0 100.0

2nd phase

-

-

9.3-10-4 8.6-10 -4 8.2. l0 -4 8 . 2 " 10 -4

-

-

4.9.10

-4

NADP binding to G6PD: fluorescence and FPLC studies Changes of the intrinsic fluorescence of proteins have been widely used to monitor ligand-protein interactions and conformational changes of enzymes [21,22]. Fig. 5 shows that NADP produced a large quenching (approx. 60%) of the intrinsic fluorescence of G6PD. In contrast, glucose 6-phosphate only quenched about 10% of the intrinsic fluorescence of G6PD (not shown). The data can be adequately fltted to a simple binding process as indicated in Materials and Methods. From these results a dissociation constant of NADP of 90 + 10/~M was derived. Fig. 5 also shows that a change of temperature from 25 to 40°C only slightly altered the dissociation constant value, thus excluding an important contribution of ionic interactions, largely enthalpic, in the formation of the NADP-G6PD complex Therefore, we concluded that NADP binding to G6PD was dominated by hydrophobic interactions, likely involving aromatic rings. Because the observed quenching of the intrinsic fluorescence was unusually large in protein-ligand interactions, and also because the time course of the inactivation by heat, Gdn-HCI and trypsin strongly suggested that isomerization between the enzyme conformations stabilized in the presence and in the absence of NADP was a slow process, we did FPLC studies aiming to check the possibility that the aggregation state of G6PD could be altered upon NADP binding. Fig. 6 shows that this is indeed the case. Micromolar NADP concentrations shifted the state of aggregation of G6PD to higher oligomeric s.tates, likely dimers and tetramers. However, it was difficult to precisely define the higher oligomeric state, due to the low resolution of the column used to resolve proteins of molecular weight above 200 kDa. In addition, the FPLC pattern showed little resolution of monomeric and dimeric forms of G6PD, while they were clearly separated from higher oligomeric forms. This result was consistent

104 A 28One

A 280 am

;;~

l

Ib

Ib

a

o.l

0,04

0.0~

0,02

1;)

;5

2'o

A260nm

rain

d

(11o-3)

A280nm

flt

~

2'o

n

b

O.O4

O.Og

A

lb

;5

2'o

rain

Fig. 6. FPLC elution patterns of NADP and bass G6PD on Superose 12. Chromatographic conditions are described under Materials and Methods. Arrows at the top of each trace indicate the retention time (t R) of the following molecular weight standards (from left to right): Ferritine, 440 kDa (t R =8.2 rain); Aldolase. 158 kDa (t R = 10.0 rain); Albumin, 67 kDa (t R = 11.7 rain); and Chymotrypsin, 25 kDa ( t R = 15.3 min). Trace a; 50/,LI sample of 80/~M NADP: trace b; 200/~l sample of G6PD. Enzymatic activity was detected in peaks l (0.6 mlU/ml), 2 (13.3 mlU/ml) and 3 (5.0 mlU/ml): trace c: 500 gi sample of G6PD incubated during 30 min at 20°C with 150/~M NADP: trace d: 500/~l sample of peak 3 from trace b incubated during 30 rain at 20°C with 150 # M NADP. G6PD activity was detected in all the fractions from the broad peak marked with an asterisk.

with a rapid equilibrium between lower oligomeric states, and a slow equilibrium between higher oligomeric states. This situation closely resembles the behaviour of another regulatory enzymes, such as glycogen phosphorylase [23], and is likely to indicate that the protein domains involved in m o n o m e r / monomer interactions were not those involved in d i m e r / d i m e r interactions. Trace d of Fig. 6 strongly supports this hypothesis, for it shows that incubation with 150/~M NADP of the protein from peak 3 (monomer) became a mixed oligomeric distribution, as revealed by reloading it again onto the FPLC column.

Discussion

The results reported in this paper clearly show that micromolar concentrations of NADP produce a conformational drift in the bass G6PD, which induces an oligomerization of the enzyme. It is to be recalled that human G6PD, the G6PD studied in deeper detail, has been reported to undergo a physical transition between two states depending on NADP concentration, each one presenting a different rate of thermal denaturation [9]. A stabilizing effect of NADP on G6PD against proteolysis in vivo was inferred [8] (e.g., a decrease of the turnover rate of this enzyme), as correlation be-

tween in vitro thermostability and in vivo turnover rate of proteins has been found [3]. From the results of Gdn-HCl and trypsin inactivation it could be concluded that G6PD exhibited two main kinetic phas~-~ of inactivation, of which the rapid phase was largely dependent upon the presence of micromolar NADP concentrations. This can be explained in terms of the coexistence in solution of two different forms of the folded state with different susceptibilities to denaturant (Gdn-HCl or trypsin), in slow equilibrium in the seconds to minutes time-scale range, and that the binding of NADP displaced the enzyme towards a more stable conformation against inactivation. On these grounds, two simple schemes could account for the effect of NADP: E + NADP ~ E. NADP ~ E " NADP

(a)

E ~ E ' + NADP ~ E'-NADP

(b)

Because the titration of the intrinsic fluorescence of G6PD with NADP did not reveal allosteric properties in the saturation process, scheme (b) appears unlikely. However, FPLC studies (Fig. 6) showed that in the absence of NADP, at protein concentrations as low as several/zg/ml, G6PD is in equilibrium between different oligomeric states. In addition, the biphasic charac-

105 teristics of G6PD inactivation by trypsin and unfolding by Gdn-HCI in the absence of added NADP is also consistent with this observation. Therefore, it is likely that both states, E and E', can oligomerize. It has been noticed earlier that G6PD from several sources is stabilized in oligomeric forms, mostly dimers [7,24]. Based on steady-state kinetic studies, we have previously suggested that bass G6PD is likely to cyclically interconvert between two conformational (dimeric) states of different affinity towards NADP and NADPH [7]. Here we present direct experimental evidence for a strong effect of NADP upon the conformation of G6PD. Moreover, the biphasic time course of Gdn-HC! unfolding of this enzyme allows to raise the conclusion that the displacement from E ' - N A D P to E is a slow kinetic process (e.g., with a rate constant lower than the rate constant of the fastest component of the unfolding process). On the contrary, there should not be a clear kinetic pattern of two populations of G6PD undergoing separate denaturation. It thus appears that NADP induces an oligomeric state of the enzyme more stable than the state predominant in the absence of this coenzyme. Moreover, it can be concluded from this analysis that the amplitude of the fast and slow phases of the unfolding process is directly proportional to the mole fraction of the enzyme in E and E ' - N A D P forms. The turnover number of the catalysis by bass G6PD is in the range of its [20], quite similar to that of human G6PD (Bautista, J.M., Mason, P.J. and Luzzatto, L., unpublished results). Therefore, during the catalysis the cyclic interconversion between E and E' is much faster than in our experimental conditions (i.e. bass G6PD shows hyperbolic kinetics in the steady state studies of initial velocity versus NADP concentration [7,20]). Because glucose 6-phosphate alone does not produce any significant effect on the time course of Gdn-HCI unfolding or trypsin inactivation, nor on thermal inactivation of G6PD, it is unlikely that glucose 6-phosphate produces a conformational drift towards the E'-conformation. Binding of both substrates, NADP and glucose 6-phosphate, appears to be required to promote a rapid and cyclic turnover between E and E' states. A similar kinetic behaviour has been demonstrated for the operation of P-ATPases [25,26], which are also energy transducing enzyme system. In these latter enzyme systems the displacement between different conformational states is coupled to the catalysis. Owing to the experimental difficulties of demonstrating this point in G6PD, it is of clear relevance to the progress of our understanding of the mechanism of catalysis, and deserves to be elucidated in future studies. A closer analysis of the apparent dissociation constants of NADP (Kd) reveals that the values derived

from the different experimental studies reported in this paper ranges between 1-2 /.tM (from trypsin inactivation) and 90 _+ 10 # M (from titration of intrinsic fluorescence), while Gdn-HCI unfolding and thermal inactivation data yield intermediate values, 50-75/~M and 1-10 # M , respectively. These values have to be compared w,;th the value of 30-33/.tM NADP derived from steady-state kinetic studies for K m and K d [20]. Because the FPLC patterns clearly show a mixed distribution of oligomeric states of G6PD, at first we have considered the possibility that the variation of the values of K d could be related to a different oligomeric state distribution of G6PD in the experimental conditions used throughout this study, assuming that NADP interacts with different affinities with different oligomeric states of G6PD. However, there is not any correlation between the value of K d and the concentration of protein u~ed in each of the experimental approaches referred to above, and this is the primary variable controlling the average aggregation state distribution of proteins. On average, the concentration of protein used in the different studies carried out ,,vas 0.2 /.~g/ml (Gdn-HCI unfolding), 1.8/.~g/ml (thermal inactivation), 27/zg/ml (trypsin inactivation) and 68/.tg/ml (fluorescence titration). Futhermore, from initial kinetic studies we have not found a strong dependence of the K m of NADP (e.g., 21 # M at 10°C and 30/.tM at 25°C [20]), nor of the K d directly estimated from fluorescence titration data (see Results). These results are consistent with the hypothesis of an interaction of NADP-G6PD dominated by the short range interactions established between the aromatic rings and amino acid side chains, basically entropically driven. A similar conclusion has been raised for glutathione reductase [27] and for triose phosphate isomerase [28]. Previous kinetic studies [7] have shown that two NADP molecules are functionally coupled during the catalysis of bass G6PD. The more simple hypothesis is that the active functional unit is a dimer, and experimental data supporting this hypothesis have been reported elsewhere [7], and in this paper (see the FPLC results). Also, human G6PD has been shown to be predominantly in the dimeric state [24]. In addition, G6PD from different sources appears to be a family of enzymes having a large homology in their amino acid composition and highly conserved functional domains [29,30]. In particular, the bass and human G6PD also show a similar kinetic behaviour towards the substrates (NADP and glucose 6-phosphate) and substrate analogues. Therefore, the results reported in this paper suggest that the stepwise NADP binding, to G6PD produces conformational changes leading to a different structural environment of the NADP binding sites, e.g., they suggest an allosteric sequential model to describe NADP binding to bass G6PD. An allosteric kinetic

106 behaviour of h u m a n G 6 P D has been r e p o r t e d u n d e r certain experimental conditions [12-14] *. This is presented in the following kinetic scheme: NADP

and S. AIgarin from C U P I M A R for providing bass. J.M.B. thanks the Universidad C o m p l u t e n s e for financial support.

NADP

References E.

" E" NADP ~.

"~ E" NADP2

where E a n d E ' are dimers, likely the dominant oligomeric state during the catalysis [12-14], with Kdl < Kd2. The presence of a high affinity binding site in bass G 6 P D suggests the existence of negative cooperativity during the catalysis, and that the value of Ko obtained from initial steady-state studies is reflecting the product average [Pt "K~ "P2"Kd2]1/2, where Pl and P2 represent the average fractional residence time of the enzyme in E a n d E ' states, respectively. Within a factor of two, e.g., within the precision range dictated by thermodynamics consideration of the thermal noise, the values of the N A D P kinetic constants ( K m and Kd), and the hyperbolic curves of activity vs. N A D P concentration obtained for bass G 6 P D [7] agree with this prediction. It has to be recalled here that the K d value for N A D P r e p o r t e d for the human G 6 P D - N A D P complex from equilibrium dialysis data, 1.5 nM [31], is largely different from the K m for N A D P , 5 - 1 3 /zM, derived from initial kinetic experiments [12-14]. On these grounds, proteolytic fragmentation and thermal unfolding leading to inactivation of G 6 P D a p p e a r s to be largely reflecting the binding of the first N A D P molecule to the enzyme, while fluorescence titration and G d n - H C l unfolding mostly monitor the binding of the second N A D P molecule.

Acknowledgments We are indebted to Professor A. G a r r i d o - P e r t i e r r a for help with the F P L C experiments and for helpful c o m m e n t s and discussions during the early stages of processing this manuscript, We thank Professor L. Luzzatto for valuable discussions and D. M c D o n a l d for help with the manuscript. Thanks are also due to L.F. Bautista in designing some of the figures for this p a p e r

* Hyperbolic behaviour of human G6PD in steady-state kinetic studies has been reported by several authors. This discrepancy with the results from authors reporting allosteric behaviour seems to arise from differences in the precise protocol adopted. In this sense the conditions for sigmoidicity in steady-state kinetic assays has been reported to be high concentration of glucose 6-phosphate and factors favouring G6PD in dimeric form [12-14]. During the time of reversion of this paper a report clarifying this point has been published (Adediran, S.A. (1991) Biochimie 73, 1211-1218).

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