Eur. J. Biochem. Y8, 425-430 (1979)

Glyceraldehyde-3-phosphate Dehydrogenase of Scenedesmus obliquus A Kinetic Analysis of the Effects of Nucleotide and Dithiothreitol on the Production of NADPH-Dependent Activity

Sandra WOODROW, Michael J. O'BRIEN, John S. EASTERBY, and Roy POWLS

Department of Biochemistry, University of Liverpool

(Received January 13, 1979)

Incubation of the hexadecameric glyceraldehyde-3-phosphate dehydrogenase of Scenedesmus obliquus with dithiothreitol causes a transient increase in NADPH-dependent activity. When NADPH is also incorporated in the incubation buffer, the induced NADPH-dependent activity is stabilised. In the presence of NADPH, the rate of induction of NADPH-dependent activity is directly propor- tional to dithiothreitol concentration. This is consistent with either a dithiothreitol-mediated reduc- tion of disulphide bonds or dithiothreitol-promoted disulphide exchange in the enzyme without prior formation of a non-covalent complex between enzyme and reductant. A kinetic analysis of the tran- sient induction of the NADPH-dependent activity by dithiothreitol alone shows that the activation is thiol-dependent with a second-order rate coefficient of 4.3 M-' min-'. In contrast, the ensuing in- activation is spontaneous and does not involve thiol. It proceeds with a first-order rate coefficient of 0.032 min-', corresponding to a half-time of 22 min. It is concluded that dissociation of the hexade- cameric enzyme to the tetramer is promoted by dithiothreitol whereas further dissociation to an inactive dimer results purely from the inherent instability of the tetramer in the absence of NADPH. The rate of induction of NADPH-dependent activity by dithiothreitol increased hyperbolically with respect to NADPH concentration, implying that NADPH has more than a stabilising role in the activation process. These observations are discussed in relation to the photoreductive process thought to occur in vivo.

The NADPH-dependent activity of the glyceral- dehyde-3-phosphate dehydrogenase of plants [I, 21 and algae [3] has been shown to be stimulated in vivo by light. Accompanying the increase in NADPH- dependent activity, that linked to NADH was depressed. Such a change in the coenzyme dependence of enzyme activity has been considered to be necessary to enable the enzyme to function in carbon fixation by the reductive pentose phosphate cycle [4]. The effect of light was reversible and could be shown to be independent of protein synthesis [5,6] .

Attempts have been made to elucidate the molec- ular basis of this type of enzyme activation. Using soni- cated chloroplasts isolated from dark-adapted spinach

Enzymes. Glyceraldehyde-3-phosphate dehydrogenase or D&C-

eraldehyde-3-phosphate: NAD(P) oxidoreductase (phosphorylat- in&) (EC 1.2.1.13); fructose 1,6-bisphosphatase or ~-fructose-1,6- bisphosphate I-phosphohydrolase (EC 3.1.3.1 1).

[7] it has been shown that incubation with dithiothrei- to1 or with NADPH stimulates the NADPH-depen- dent glyceraldehyde-3-phosphate dehydrogenase ac- tivity. Moreover, the effects of dithiothreitol and NADPH were additive: together they caused an activation identical in extent to that of optimal illumination in vivo. It was suggested that dithio- threitol-mediated reduction of disulphide bridges to sulphydryl groups in the enzyme protein was ac- companied by a specific action of NADPH in the activation process [7].

Purified enzyme from spinach chloroplasts was shown by Pupillo and Piccari [8] to undergo stimula- tion of the NADPH-dependent activity when in- cubated with dithiothreitol. However in this case in- cubation with dithiothreitol and NADPH was less effective than incubation with dithiothreitol alone. In contrast to the rapid activation seen in chloroplast

426 Glyceraldehyde 3-phosphate Dehydrogenase of S. ob/iyuus

homogenates [4], these thiol-induced activations were very slow processes and required up to five hours for completion [8]. The activation of the NADPH-depen- dent activity was associated with depolymerization [S].

Similar changes in coenzyme dependence of gly- ceraldehyde-3-phosphate dehydrogenase activity mo- dulated by light have been seen in algae [3]. A hexade- cameric form of the enzyme has been purified from the green alga Scenedesmus obliquus and its coenzyme dependence can be modified in vitro [9- 1 I]. The enzyme is predominantly active with NADH as COfac- tor, but can be converted into a tetrameric form char- acterised by activity principally linked to NADPH. Such a change in coenzyme requirement of activity is produced by incubation of the enzyme with either 1,3-bisphosphogIycerate [lo] or dithiothreitol and NADPH [ l l ] . The algal enzyme differs from the spinach chloroplast enzyme in that incubation with dithiothreitol alone causes a marked induction of NADPH-dependent activity. However, such stimula- tion is only of a transient nature; prolonged incuba- tion causes further dissociation of the enzyme to an inactive dimer [l I]. This inactivation on prolonged incubation with dithiothreitol might seriously ques- tion the widely-held view that thiol-dependent activa- tion of the isolated enzyme is analogous to the light- dependent stimulation of NADPH-dependent activity seen in vivo.

In an attempt to clarify the molecular basis of light modulation of enzyme activity in vivo, we have made a detailed kinetic study of the promotion of algal NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase by dithiothreitol in vituo, in the pre- sence and absence of NADPH.

METHODS

Culture Conditions

Scenedesmus obliquus (Cambridge 276/6a) was grown heterotrophically on glucose as described by Kessler et al. [12].

Enzyme Purification, Assay and Activation

The hexadecameric form (D enzyme) and tetra- meric form (T enzyme) of S. obliquus glyceraldehyde-3- phosphate dehydrogenase were purified and assayed as described previously [lo]. For activation the purified enzyme (0.1 unit of NADPH-dependent activity) was incubated at 30 "C with the compounds specified in 1 ml 85 mM Tris-HC1 pH 7.5. At specified times 0.05-ml aliquots were removed from the incubation mixture and added to the assay mixture to a final volume of 1 ml. An enzyme unit is defined as the quantity of enzyme transforming one micromole of substrate per minute at 30 'C.

RESULTS

Incubation of the hexadecameric form of glyceral- dehyde-3-phosphate dehydrogenase from S. ohliquus with dithiothreitol and NADPH caused stimulation of the NADPH-dependent activity. The effect of varying the dithiothreitol concentration whilst the NADPH concentration was fixed at 1.3 mM is pre- sented in Fig. 1. The rate of increase in NADPH- dependent activity was progressively raised by the increasing dithiothreitol concentration and under these conditions the initial rate of promotion of NADPH-dependent activity was first order with respect to dithiothreitol concentration (Fig. 2). The effect of NADPH concentration on the promotion of NADPH-dependent activity at fixed dithiothreitol concentration (18 mM) is shown in Fig. 3. The rate of induction of activity increased hyperbolically with respect to NADPH concentration as shown in Fig.4, and the dissociation constant for the NADPH-enzyme complex derived from this figure was 18 FM.

Inclusion of NADPH in the incubation mixture had two effects on NADPH-dependent activity: it increased the rate of promotion and stabilised it (Fig. 3). By contrast, prolonged exposure to dithio- threitol alone results in initial promotion followed by a decline in the NADPW-dependent activity. The time courses of these changes at various dithiothreitol concentrations are presented in Fig. 5. Increasing the concentration of dithiothreitol increased the rate of induction and the maximum NADPH-dependent activity.

I 1 I I I I I + .~ .- c " m

0 10 20 30 40 50 60 Time (min)

Fig. 1 . The $ecf o f i ,~cuhu~ion of S. obhquus g!,.c.c~l.rr/dcl1~tl~~-3-phos- pllatr dehydrogennsr ivirlz vurying conc~mtrations of dit lziotlwitd in tkepi~c~sence OfNADPH. The hexadecameric form of the enzyme was incubated (as described under Methods) with various concentrations of dithiothreitol in the presence of 1.3 mM NADPH and the time course of changes in the NADPH-dependent activity determined. The concentrations of dithiothreitol used were: ImM (A ~- -A), 2.7mM(. ~ - 0 ) , 4 . 5 m M ( t - ~ ~ . ) . 9 . 1 mM(a--a). 18.2mM (0- --0) and 46 mM (u- ~ u)

S. Woodrow, M. J . O'Brien, J. S. Easterby, and R. Powls

-- 1.0 -

.L 0.0-

X 0.6 - P <m - 0.4 -

c .- E

0

E

. c

0.2 -

4

427

I I

10 I I I I I

[Oithiothreitol] (mM)

Fig. 2. Tlir relations/iip betiiwn the rate os induction of NADPH- dependent gl~ceraldehyde-3-phosphate dehydrogenase activity and ciitiiiotlireitol concerztration. The initial rate of induction of NADPH- dependent activity at the various concentrations of dithiothreitol was obtained from Fig. 1. The linearity of the plot shows the rate of induction of NADPH-dependent activity to be first order in dithiothreitol concentration

I I I I I I 1

100 t J Y

I I I I I 1 I 0 5 10 15 20 25 30

0'

Time (mini

Fig. 3. Thc~ c.ffect (f incubation 1 f S . obliquus g l ~ c e r . a l d ~ ~ / i ) ~ ~ ~ e - 3 - ~ ~ l i o ~ - phute cleli).drogenase with varying concentrations of NADPH in the presence of dithiotlireitol. The hexadecameric form of the enzyme was incubated (as described in Methods) with various conccntra- tions of NADPH in the presence of 20 mM dithiothreitol. The time course of the resulting changes in NADPH-dependent activity was compared with that observed when the enzyme was incubated with dithiothreitol alone. The concentrations of NADPH in the incuba- tion buffer were: 0 (A-A), 3 pM (I----.), 6 pM (0 -o), 10 pM (A --A), 60 pM (-0) and 300 pM (-- 0)

The transient nature of the stimulation of activity can be considered to result from the sum of two effects [l I]. Initially, the hexadecameric enzyme is depolymerised to a tetrameric species (T' form) ac- companied by an increase in NADPH-dependent activity at the expense of activity linked to NADH. The decline in NADPH-dependent activity observed on prolonged incubation with dithiothreitol results

,

IIINADPH] (pM-')

Fig.4. The relationship between the rate of induction oJ' R'ADPH- dependent glyceraldehyde-3-phospl1ate dehydrogenase activity and NADPH concentration. The initial rates of induction of NADPH- dependent activity were obtained from the data presented in Fig. 3. The rate of induction of NADPH-dependent activity promoted by dithiothreitol and NADPH ( r a ) was corrected for that promoted by dithiothreitol alone ( r b ) . The linearity of the double-reciprocal plot demonstrates the hyperbolic nature of the binding curve for NADPH and a value of 18 pM is obtained for the dissociation constant of the enzyme-NADPH complex

120

80

40

n -0 10 20 30 40 50

Incubation time Irnin)

Fig. 5. The eJJkct of dithiothreitol concentration on the tirnr ~01~r.se of changes in NADPH-dependent activity of S . obliquus gi.vcrral- d~hyde-3-phosphate dehydrogenase. The concentrations of dithio- threitol used in the incubations were (A) lOmM, (A) 20mM. (0) 30 mM, (0) 40 mM, (0) 60 mM, (I) 20 mM plus 1.3 mM NADPH

from the further depolymerisation of the tetramer into an inactive dimer. This may be described by the scheme in Fig. 6 in which two consecutive processes, each first order in enzyme, are envisaged to occur. It follows from such a mechanism that the concentra- tion of the tetramer at a particular time (and hence the NADPH-dependent activity) will depend upon the values of kl and kz, the rate coefficients for the two

428 Glyceraldehyde 3-phosphate Dehydrogenase of S. obliquus

NADPH NADPH T'

hexadecamer tetramer inactive

/I Dimer dithiothreitol

kl k2

NADH-dependent NADPH-dependent

Fig. 6. Schenie,for the changes in gl.~ceraldehyde-3-phosphate dehq'dro- genase activity induced by dithiothreitol

consecutive reactions. Inclusion of NADPH with dithiothreitol in the incubation buffer stabilizes the induced NADPH-dependent activity [l I ] and the nucleotide has been considered to bind to the enzyme preventing further depolymerization of the tetramer (T'). The effect of NADPH is thus to reduce k2 and possibly block this step completely when present at saturating concentration. However, it is apparent from Fig.5 that NADPH also has an effect upon kl since the rate of induction of NADPH-dependent activity elicited by incubation with 18 mM dithiothrei- to1 was doubled when 1.3 mM NADPH was also in- cluded in the incubation buffer.

The scheme shown in Fig. 6 results in the following equations describing the time course of changes in the concentrations of the D and T' forms of the enzyme. A similar kinetic approach has been used in the study of the glyoxylate carboligase of Escherichiu coli [I 31.

[D] = [D]o. e-"" (1 1

1 ~ ln f* = - k2

1 k2 ~- (In A* - In Amax) . (4)

Here the concentrations are expressed on a mass rather than molar basis. [D]o is the initial concentration of the D form of the enzyme, ,f* is the fraction of the enzyme present as the T' form at the maximum in the time course of NADPH-dependent activity and t* is the time corresponding to maximal activity. A* re- presents the NADPH-dependent activity and A,,, is the theoretical maximum NADPH-dependent activity which would be attained if all the enzyme was present as the T' form. To analyse the data of Fig. 5 , In A * was plotted against t * for each dithiothreitol concentration studied and k2 was determined from the slope of the plot. The ordinate intercept gave A,,,, f * was cal- culated as the ratio of A* to A,,, and used in conjunc- tion with Eqn ( 3 ) (Fig.7) and the derived value of k2 to find k l . This method for the separation of k l and k2 suffers from inaccuracies at kl/k2 ratios greater than 5 owing to the insensitivity of j * to the kl/k2 ratio in this region (Fig. 7). The values obtained were checked by computer simulation of the equations.

0.2 tf 0' I ' " ' " ' ' '

kl /k2

0 2 4 6 8 10 12 14 16 18 20 22

Fig. I . Dependence of' the nzuximunl fraction qf enzjmc present as the T'form upon the k l / k z ratio. The graph was derived from Equa- tion (3)

150 7

Fig. 8. The relationship betit.een the NADPH-tlependent activitj qf the enzyme at the turning point in the activity/time curve (A*) and the time at which this acrivity w a s reached (t*). Each data point represents a complete experiment at a particular dithiothreitol con- centration and is derived from Fig. 1. Activity is plotted on a log scale according to Equation (4). The slope of the plot gave a k z value of 0.032 min-' corresponding to a half-time of 22 min for the in- activation. A,,, is 146 pM min-'

The value of kz determined in this way (Fig.8) was 0.032 min-', corresponding to a half-time for the inactivation of the T' form of the enzyme of 22 min. The linearity of the plot in Fig. 8 testifies to the absence of any effect of dithiothreitol on kz. The kl values obtained for incubation at various concentrations of dithiothreitol are shown in Fig. 9 and gave a value of 4.3 M-' min-' for the second-order rate coefficient controlling the dithiothreitol-promoted formation of the T' form of the enzyme.

The activity of the naturally occurring tetramer (T form) of S. obliquus glyceraldehyde-3-phosphate dehydrogenase is principally linked to NADPH, although it also possesses some NADH-dependent

S. Woodrow, M. J. O'Brien, J . S. Easterby, and R. Powls 429

I I I I I I I I

0.23 0'251 0.05 t I' 1

o v I I I I 1 I I 0 10 70 30 40 50 60 70

(Dithiothreitol] (mM)

Fig. 9. The tlependence of the pseudo7ftst-order rate coefficient f;)r the gencmtion of NADPH-dependent activity on dithiothreitol con- centration. The derived second-order coefficient was 4.3 M- ' min-l

I 1 I I I I l a'

, 0 10 20 30 40 50 M) 70

Time (rnin)

Fig. 10. The irzactivatiori of the tetrameric glyc~~raldehyde-3-phosphote dehydrogenase of S . obliquus by dithiothreitol and the Cflect of NADPH. The tetrameric enzyme (T form) was incubated with 18 mM dithiothreitol and 1.3 m M NADPH; the resulting NADPH (L1- -0) and NADH (0 -0) dependent glyceraldehyde-3- phosphate dehydrogenase activities are presented. Incubation with 18 mM dithiothreitol alone caused a decrease in activity linked to both NADPH ( b W ) and NADH (O--O). After a 16-min incubation with dithiothreitol, a portion was removed and made

.3 mM in NADPH. This treatment immediately arrested the in- activation of NADPH-dependent activity (A--A)

activity. On incubation of this form of the enzyme with dithiothreitol (1 8 mM), activity dependent upon either NADPH or NADH was progressively lost (Fig. 10). This dithiothreitol-induced decline in ac- tivity was prevented by the addition of NADPH (1.3 mM) to the incubation buffer. The dithiothreitol- promoted inactivation was shown to be associated with the formation of increasing amounts of a species with a sedimentation coefficient of 4.5 S. This is very similar to the inactive dimer formed on prolonged incubation of the hexadecameric form of the enzyme with dithiothreitol [I 11.

DISCUSSION The behaviour of dithiothreitol in stimulating the

activity of a number of enzymes has often been con- sidered to be analogous to that of a chloroplast reductant photo-produced in vivo [l 31. Both com- pounds could alter the conformations of enzymes by the reduction of intersubunit or intrasubunit disul- phide bonds, by reduction of sulphenic acids [15] or by reductive removal of a protein modifier. Changes of enzyme activity might accompany such conforma- tion changes and this has been demonstrated for spinach chloroplast fructose 1,6-bisphosphatase. In this case the enzyme activity is affected by incubation with dithiothreitol and there is a parallel increase in the number of free sulphydryl groups [16,17]. How- ever, no change in the molecular weight occurs [14]. In glyceraldehyde-3-phosphate dehydrogenase of spinach chloroplasts [8] and S. ohliquus [ I l l depoly- merization results from dithiothreitol-promoted con- formational transitions. The first-order kinetic be- haviour, with respect to dithiothreitol, seen in the induction of NADPH-dependent activity of the algal enzyme (Fig. 2), indicates that binding of dithiothreitol to the enzyme to form a non-covalent complex does not precede activation. This finding is consistent with simple reduction of disulphide bonds but might also be expected if dithiothreitol promoted disulphide ex- change which resulted in changes in protein conforma- tion.

It has been suggested that photosynthetic electron flow is responsible for the formation of a component analogous in its action to that of dithiothreitol [I7 - 191. However, the inactivation of algal glyceral- dehyde-3-phosphate dehydrogenase on prolonged ex- posure to dithiothreitol was seen as a problem if di- thiothreitol-dependent activation was truly analogous to the stimulation of NADPH-dependent activity in vivo. The present study shows that the further dis- sociation of the T' form of the enzyme to an inactive dimer is independent of thiol and therefore not directly promoted by dithiothreitol. All pyridine nucleotides so far studied are capable of stabilizing the activity of the T' form of the enzyme [ll]. In the case of NADPH, the binding of this nucleotide presumably causes a conformational change in the enzyme which also makes it more susceptible to reaction with dithiothreitol. This results in the promotion of the T' form of the enzyme. Binding of NADPH also stabilizes the T' form against further depolymerization to an inactive dimer. The binding of NADPH to the enzyme is of a hyperbolic nature (Fig.4) and the dis- sociation constant of the enzyme-NADPH complex involved is very similar to the reported Michaelis constants of the algal enzymes (D and T forms) [9]. This suggests that all functions of NADPH described here are attributable to the binding of the nucleotide to the catalytic site.

430 S. Woodrow, M. J. O’Brien, J . S. Easterby, and R. Powls: Glyceraldehyde 3-phosphate Dehydrogenase of S. ohliquus

Other compounds present in illuminated chloro- plasts are also effective stabilizers of T’ enzyme ac- tivity. These include ATP [20], inorganic phosphate and 1,3-bisphosphoglycerate [lo]. It appears that most, if not all, metabolites which bind to the enzyme offer a degree of stabilization. Consequently, if the light-mediated stimulation of NADPH-dependent activity in vivo occurs by a process similar to dithio- threitol stimulation, adequate metabolites will be present to stabilize the tetramer against inactivation. However, it is also possible that reduction occurs in vivo with greater specificity than in vitro where dithiothreitol could over-reduce the enzyme to produce a tetramer which is inherently unstable. With regard to this, two other factors merit comment. The maximum NADPH-dependent activity is observed on incubation with dithiothreitol in the presence of NADPH (Fig. 1) and is higher (193 pM min-’) than the A,,, value estimated from Fig. 3 (146 pM min-’). It therefore appears that NADPH can promote NADPH-depen- dent activity in excess of that attributable to dithio- threitol. Secondly, in the presence of dithiothreitol the initial rate of generation of NADPH-dependent activity is stimulated by NADPH (Fig. 5). A possible explanation of these effects is that over-reduction of the enzyme occurs with dithiothreitol compared with the reductive process in vivo. NADPH could serve to confer a degree of specificity on the reduction, possibly by protecting specific disulphide bonds. In the presence of NADPH a species more closely related to the native NADPH-dependent enzyme (T form) might be gener- ated. If this is the case one might expect the T form of the enzyme itself to be susceptible to dithiothreitol- promoted inactivation. This was indeed observed (Fig. 10) and an ultracentrifugation study indicated that depolymerisation to the dimer occurred. It may therefore be unnecessary to propose any components other than the simple ligands encountered in the chloroplast to confer specificity on the process in vivo. It is also possible that the activation process here represented as being controlled by the single rate coefficient kl may in fact involve reduction of more than one category of disulphide bond on the protein.

The findings reported in the present paper empha- sise the importance of a reductant in the stimulation of the NADPH-dependent glyceraldehyde-3-phos- phate dehydrogenase activity of S. obliquus. It is only in the presence of such a compound that NADPH formed by photosynthetic electron transfer can sig- nificantly stimulate the NADPH-dependent activity of the algal enzyme. Indeed the greatest stimulation of the NADPH-dependent activity in vitro has been seen when incubation with dithiothreitol is accom- panied by either NADPH or 1,3-bisphosphoglycerate

[lo, 111. During photosynthesis the latter compound is formed from 3-phosphoglycerate at the expense of ATP synthesised by photophosphorylation.

The identity of the photo-produced chloroplast component necessary for enzyme activation is still not certain. One possibility is that reduced ferredoxin passes reducing equivalents to thioredoxin which then serves as the direct reductant of light-activatable enzymes [17]. Such a system has been reported to be involved in the light-dependent activation of spinach chloroplast glyceraldehyde-3-phosphate dehydrogen- ase [21,22]. The action of dithiothreitol on this enzyme has been suggested to be mediated by enzyme-bound thioredoxin [22]. Other possible mechanisms for the activation of these enzymes in vivo should not, however, be disregarded. Light-induced conformational chan- ges of chloroplast membranes are known to result in the exposure of vicinal dithiols to the stroma [19] and it is possible that these could be involved in modulation of activity of chloroplast enzymes.

We wish to thank the Science Research Council for the award of Research Studentships to Sandra Woodrow and M. J. O’Brien, and Mrs J. D. Baker for technical assistance.

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S. Woodrow, M. J. O’Brien, J. S. Easterby, and R. Powls, Department of Biochemistry, University of Liverpool, P. 0. Box 147, Liverpool, Great Britain, L69 3BX