glucose 6-phosphate dehydrogenase from · glucose 6-phosphate dehydrogenase from leuconosfoc...

THE JOURNAL OF Ihmcmx~. CHEMISTRY

Vol. 246, No. 7, Issue of April 10, pp. 2047-2057, 1971

Printed in U.S.A.

Glucose 6-Phosphate Dehydrogenase from

Leuconostoc mesenteroides

KINETIC STUDIES*

(Received for publication, October 5, 1970)

CHARLES OLIVE,~ MARY E. GEROCH, AND H. RICHARD LEVY

From the Biological Research Laboratories, Department of Biology, Syracuse University, Syracuse, New York

13210

SUMMARY

Glucose 6-phosphate dehydrogenase from Leuconosfoc mesenferoides catalyzes the oxidation of glucose 6-phosphate by either NADP+ or NAD+. Steady state initial velocity studies and product inhibition studies using NADPH were conducted for the NADP-linked reaction catalyzed by this enzyme. The data were consistent with a simple, ordered, sequential mechanism for this reaction in which NADP+ is bound first to the enzyme and NADPH released last. Kinetic studies of the NAD-linked reaction indicated a more complex mechanism. Initial velocity studies, product, and alternate product inhibitions using NADH and NADPH, respectively, and alternate substrate studies using either NAD+ and NADP+ with glucose 6-phosphate, or glucose 6-phosphate and 2-deoxyglucose 6-phosphate with NAD+, suggested an ordered, sequential mechanism with isomerization of free enzyme. The enzyme form which binds NADH is proposed to be the same form binding NADPf and NADPH; NADf is assumed to bind to an isomeric form.

A detailed study of the effect of varying pH from 5.74 to 9.90 was carried out for the NAD-linked reaction. From the changes in kinetic constants with pH some tentative suggestions emerged concerning enzyme groups which may participate in the binding of NADf and glucose B-phosphate. Among these, the most clear-cut was the participation of a group on the enzyme with pK’ = 8.9 in binding of glucose 6-phosphate. Evidence was obtained that this group is not a cysteine. Further evidence, using pyridoxal 5’-phosphate, suggested that the group may be the c-NH2 group of a lysine.

Glucose 6-phosphate dehydrogenases can be distinguished on the basis of their nucleotide specificity. One group, exemplified by the enzymes from brewers’ yeast (1) Candida utilis (2) and

* This investigation was supported by Grant AM07720 from the United States Public Health Service.

$ Predoctoral Trainee in Microbiology, National Institutes of Health. Part of this work is taken from a dissertation submitted to the Graduate School of Syracuse University in partial fulfill- ment of the requirements for the Ph.D. degree.

Escherichia coli (3) reacts exclusively with NADP+. A second group, which appears to include animal glucose 6-phosphate dehydrogenases in general (4), reacts with NADP+ but also displays weak activity with NAD+ and some of its analogues. This NAD-linked activity is not thought to have any physio- logical significance, but investigations of rat mammary glucose 6-phosphate dehydrogenase have shown that the activities with NAD+ and NADP+ respond differently to various reagents and conditions (5-7). We have presented evidence that this differential response results from alterations in the equilibrium between two enzyme forms (presumably conformational isomers) each of which reacts preferentially with one of the coenzymes. The third group of glucose 6-phosphate dehydrogenases can react approximately equally well with NAD+ and NADPf. This class includes the enzymes from Leuconostoc mesenteroides (8), Pseudomonas aerugirwsa (9), Hydrogenomonas H 16 (10) and Thiobacillus ferrooxidans.l It will be of interest to compare these three classes of enzymes for differences in their catalytic and regulatory mechanisms and to relate these to differences in structure at their active and regulatory sites.

Toward the realization of these goals we have undertaken a study of the kinetic mechanism of the reaction catalyzed by L. mesenteroides glucose 6-phosphate dehydrogenase. These studies are the subject of this communication. The isolation and some properties of the crystalline enzyme were reported previously (11). Some physical characteristics of the enzyme are described in an accompanying paper (12).

EXPERIMENTAL PROCEDURE

Standard assays (except for kinetic studies; see below) were performed in a Zeiss PM& II spectrophotometer with the thermostated cell compartment maintained at 25”. Assay mixtures (3.00-ml final volume) contained the following components: 33.0 mM Tris-HCl, pH 7.8; 3.30 mM glucose 6-phosphate; and either 0.160 mM NADPf or 2.50 mM NAD+ (neutralized to pH 7). Unless noted to the contrary, reactions were initiated with either enzyme or glucose 6-phosphate and followed by noting the increase in absorbance at 340 nm with time.

Protein concentrations were determined from the absorbances at 280 nm and 260 nm (13) or from the extinction coefficient at 280 nm after this was determined.

1 R. Tabita, personal communication.

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2048 Kinetics of Glucose 6-Phosphate Dehydrogenase Vol. 246, No. 7

The enzyme was purified as described previously (11). Some experiments were performed with enzyme subsequently purchased from Worthington Biochemical Corporation. The commercial enzyme, which was approximately 50% pure, was passed through a hydroxylapatite column (11) which removed contaminants to yield a preparation with a specific activity suffi- ciently high to permit crystallization.

The steady state kinetic experiments were performed in a Gilford model 240 spectrophotometer with a thermostated cell compartment maintained at 25” and with a built-in scale ex- pansion device, coupled to either a Leeds and Northrup Speedo- max G or a Sargent SRI, variable speed recorder. Kinetic studies of the NADP-linked reaction in which the concentration of NADP+ was varied necessitated the use of cuvettes with 50.mm light paths and a specially constructed thermostated cell compartment? which accommodated these larger cuvettes. All nucleotide solutions were prepared and, if necessary, neutralized, within 24 hours before they were to be used. The pH was checked on the day of the experiment on solutions equilibrated at 25” and after assay in cuvettes containing the largest initial amounts of NAIF and NADP+. In all experiments the exact concentrations of the solutions of glucose B-phosphate and NAD+ or NADP+ were determined enzymatically using glucose 6-phosphate dehydrogenase from either yeast or L. mesenteroides. Control experiments established that the enzyme was stable in all the buffers used for the time required to do the assays.

For the series of experiments conducted at various pH values, the appropriate enzyme dilution was assayed on the day of the experiment under standard enzyme assay conditions (see above) using NADP+ as coenzyme so that the values for V obtained in all the experiments could be compared in pH plots. The concentration of enzyme used never exceeded 10e4 times the concentration of either substrate, fulfilling steady state kinetic requirements (14). Care was taken to ensure that initial rates were being measured in all experiments.

It was necessary to determine approximate Km values for each substrate prior to an initial velocity experiment. This allowed experiments to be conducted by varying substrate concentrations over a specific range relative to each K,, e.g. 0.5 to 5 K, (15). All assays were done in duplicate and the assays at the two lowest levels, both of varied and changing-fixed substrate, were done in triplicate. Data from these initial velocity experiments were first plotted as Lineweaver-Burk plots. In all instances except possibly one (see below) linear plots were obtained. The data were then fitted to Equation 1 with the aid of a IBM System/360 model 50 computer. When data were found to conform to an ordered sequential mechanism (16) they were fitted to Equation 2

vs

v=KfS (1)

VAB

in w-hich B and H are the first and second substrates, respectively, to add to the enzyme; KiA is an inhibitor constant for A which represents the dissociation constant for the EA complex in simple, ordered sequential mechanisms (16) ; and Ka and K, are Michaelis constants for A and B, respectively. The slopes (K/V) and intercepts (I/B) obtained from the analysis of the

2 This was built by Gilford Instrument Laboratories, Inc.

data by Equation 1 were then plotted against the reciprocal concentration of the nonvaried substrate or the inhibitor concentration. The data were fitted to a straight line function using the weighting factors obtained from the first analysis. Programs for Equations 1 and 2 and for the straight line function were supplied by Cleland (17). In all the figures depicting kinetic data the lines are drawn from values supplied by the appropriate computer program.

i2~oterials-~411 nicotinamide adenine nucleotides, glucose B-phosphate, 2-deoxyglucose 6-phosphate, galactose B-phosphate (substantially glucose free), rabbit muscle lactic dehydrogenase, yeast alcohol dehydrogenase, p-hydroxymercuribenzoate, iodoacetate, and iodoacetamide were obtained from Sigma. Pyri- doxal 5’-phosphate, pyridoxal, and pyridoxamine 5’-phosphate were products of Nutritional Biochemicals. Glucose B-sulfate was synthesized and kindly supplied by Dr. I). I‘. Steiner, the University of Chicago. Yeast glucose 6-phosphate dchydrogen- ase, analytical grade, was obtained from C. E‘. Boehringer and Sons. Ammediol (2-amino-2-methyl-l, 3-propanediol) and imidazole were purchased from Eastman Organic Chemicals. Am mediol was recrystallized from ethanol and dried in a vacuum before use. All other materials used were of reagent grade.

Purified NAD+ was used in all steady state kinetic experiments. It was purified by the method of Winer (18) except that the final crystallization was omitted. Purity of the NAlF was checked by noting the absorbance at 260 nm and, after reduction with acetaldehyde and alcohol dehydrogenase, at 340 nm. NAD+ of identical purity was also purchased from Sigma.

The NADH used as product inhibitor in early experiments was purified by DEAE-cellulose column chromatography using a linear gradient of Tris-HCl, pH 7.5, between 0.005 M and 0.12 M with respect to Tris. When NADH began to emerge from the column the gradient elution was discontinued and 0.12 &I Tris- HCl, pH 7.5, was used to elute NADH in the smallest possible volume. The concentration of Tris in the purified NADII solution was determined using conductivity measurements with a Radiometer conductivity meter and a standard curve of conductivity as a function of Tris concentration. Each set of assays in which purified NADH was used was corrected so that Tris concentration was not a variable. NADH was assayed by its absorptiou at 260 nm and 340 nm using the appropriate molar extinction coefficients. Residual absorbance at 340 nm was determined on the purified NADH using excess liyruvate and lactic dehydrogenase to oxidize all NADH to NAT)+. The NADH thus obtained was pure by these criteria (19). NADH was found to be stable for at least 48 hours, according to the above criteria, when a dilute (1 nlM) solution was stored at O-4”. The purified NADH was used only in the inhibition experiments in which the fixed substrate concentration was below saturation. It was subsequently shown that commercial N;BDIT gave the same results as the purified preparation, and in later experiments employing NADH or NADPII, commercial preparations were used. Both NADH and NADPH were routinely tested for the presence of oxidized coenzyme and, if necessary, incubated with enzyme and glucose 6-phosphate to effect complete reduction before initiating the reaction with NAD+ or NAnI’+.

RESULTS

Kinetic Studies of NADP-linked Reaction

Initial Velocities-The results of an initial velocity experiment performed at pH 7.2 are illustrated in Fig. 1, 3 and L3. The


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Issue of April 10, 1971 C. Olive, hf. E. Geroch, and H. R. Levy 2049

IO

-t6 -

4

2

I/(NADP+) I/mM

C I I I

100 200 300 I/(NADP? I/mM

D

IO I/~GGP:~,mM

30

FIG. 1. Double reciprocal plots for the NADP-linked reaction. A, initial velocity ~J~IXL~ NADPf concentration at various constant levels of glucose B-phosphate. The buffer was Tris-maleate, 0.033 M (with respect to Tris), pH 7.20. The concentrations of glucose B-phosphate used were 1, 0.49 mM; 2, 0.16 mM; 3, 0.098 mivr; 4, 0.065 InM; 5, 0.049 mM. B, initial velocity versus glucose &phosphate concentrations at various constant levels of NADP+. Buffer was as in A. The concentrations of NADP+ used were 1, 58 PM; 2, 19 pM; 3, 12 pM; 4, 7.7 PM; 5, 5.8 PM. C, NADPH inhibition at saturating glucose B-phosphate concentration (3.3 mM) and varying NADP+ concentrations. Tris-HCI buffer, 0.033 M, pH 7.8, was used. The concentrations of NADPH used were 1, 0; 2, 17 pM; 3, 34 pM; 4, 51 PM; 5, 68 PM. D, NADPH inhibition at saturating NADPI- concentration (0.189 mM) and varying glucose 6-phosphate concentrations. Buffer was as in C. The concentrations of NADPH used were 1, 0; 2, 16 pM; 3,

32 PM; 4, 48 PM; 5, 64 PM.

intersecting patterns are consistent with a sequential mechanism for the NADP-linked reaction (16). The replots for slopes and intercepts from both of these patterns were linear.

Product Inhibition-Product inhibition studies were conducted with NADPH. When NADP+ was the varied substrate, linear competitive inhibition was obtained (Fig. 1C). When glucose B-phosphate was the varied substrate the inhibition was

linear noncompetitive (Fig. ID). These data are consistent with an ordered sequential mechanism for the NADP-linked reaction. The dissociation constant for NADPH was calculated from the slope replot from Fig. 1C and is given in Table I.

Analysis of the initial velocity data using Equation 2 yielded K, va.lues for KADP+ and glucose &phosphate and the dissociation constant for XADP+ (Table I).

TABLE I

Kinetic constants All values were determined in Tris-HCl at pH 7.8 except for

the Km values for both substrates in the NADP-linked reaction for which the pH was 7.2.

Substrate

NADP+ . . Glucose-6-Pa. _ . NADPH . . . . . NAD+. Glucose-6-Pg.

PM lJ‘+J 5.69 5.03

81.0 37.6

106 52.7

a Values determined in the NADP-linked reaction. * Values determined in the NAD-linked reaction.

Kinetic Studies of NAD-linked Reaction

Initial Velocities-Initial velocity studies were conducted at pH 7.8. Linear, intersecting patterns were obtained which gave linear replots. In contrast to the NADP-linked reaction, the intersection point was above the abscissa. The calculated kinetic constants are given in Table I.

Product Inhibition-Product inhibition studies with NADH were conducted by following absorbance changes at 366 nm.3 With NAD+ as the varied substrate and glucose B-phosphate, nonsaturating, linear noncompetitive inhibition was observed (Fig. 2A). When an experiment was performed using saturating glucose B-phosphate, the inhibition again appeared to be noncompetitive (Fig. 2B). When glucose B-phosphate was the varied substrate inhibition by NADH appeared to be noncom-

petitive whether NAD+ was nonsaturating (Fig. 2C) or saturating (Fig. 20).

Alternate Product Inhibition-NADPH was tested as an alternate product inhibitor for the NAD-linked reaction. With glucose B-phosphate saturating and NAD+ varied the inhibition was linear noncompetitive (Fig. 3).

Alternate Substrates-Wong and Hanes (20; see also Reference 14) suggested a method to establish the order of addition of substrates to an enzyme catalyzing an ordered sequential reaction. The basis of this technique lies in the fact that if a mixture of the first substrate to bind and one of its reactive analogues is used, the reaction mechanism then becomes second degree because the second substrate has two binary enzyme complexes with which to react. On the other hand, the mechanism remains first degree if a mixture of the second substrate to bind and one of its reactive analogues is used. Second degree mechanisms give curved reciprocal plots, whereas the reciprocal plots from first degree mechanisms are linear. The degree of curvature depends on the relative concentrations of the two binary enzyme complexes and is greatest when these are identical.

3 During the course of these studies it was found that substantial errors were introduced at high NADH concentrations. These could be attributed to the large slit widths employed. When the slit was opened more than 0.6 mm the absorbance per pmole of NADH added progressively decreased with increasing slit width. No such discrepancy was observed at 340 nm using slit widths up to at least 1.8 mm. Although a calibration procedure was devised to correct these discrepancies at 366 nm, routinely we avoided using NADH concentrations requiring slit openings greater than 0.6 mm. Failure to take this discrepancy into account initially gave results which yielded curved replots for intercepts or slopes ser.sus NADH concentration.


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2050 Kinetics of Glucose. 6-Phosphate Dehydrogenase Vol. 246, No. 7

80 - 160

60

< -

40

2 .

l I

/- B

IO 20 30

I/INAD+) I/mM

-120

-t -

-80

-40

L

D

I I IO

I,(G6?l/mM 30

-80

-60

3

-40

-20

FIG. 2. Douple reciprocal plot showing product inhibition by NADH of NAD-linked reaction. A, nonsaturating concentration of glucose 6-phosphate (0.21 mM) and various concentrations of NAD+. The buffer was Tris-HCI, 0.033 M (with respect to Tris), pH 7.80. The concentrations of NADH used were 1, 0; 2, 0.15 mM; S, 0.30 rnMn; 4, 0.45 miw; 5, 0.60 mM. Velocities were determined at 366 nm. B, saturating concentration of glucose 6-phosphate (3.3 mM) and various concentrations of NAD+. Buffers were as in A. The concentrations of NADH used were 1, 0; 2, 0.5 m&r. Velocities were determined at 366 nm. C, nonsaturating concentration of NAD+ (0.33 mM) and various concentrations of glucose B-phosphate. Buffer was as in A. The concentrations of NADH used were 1, 0; 2, 0.15 mM; S, 0.30 mrvr. Velocities were determined at 366 nm. D, saturating concentration of NAD+ (6.0 mM) and various concentrations of glucose R-phosphate. Bnffer was as in A. The concentrations of NADH used were 1, 0; 2, 0.24 mM; S, 0.51 mM. Velocities were determined at 366 nm.

In order to employ this method it was necessary to determine apparent Michaelis constants for substrate analogues. At pH 7.8 the following apparent Michaelis constants were found using NAD+ at 10 K,: 2-deoxyglucose B-phosphate, 12 mM; galactose B-phosphate, 10 mM; glucose 6-sulfate, 50 mM. The maximum velocity for each of these analogues was less than 0.3 V for glucose B-phosphate. The K, values for glucose 6- phosphate, NAD+, and NADP+ are given in Table I. The apparent K, for thionicotinamide adenine dinucleotide, using 10 K, glucose 6-phosphate, was found to be 11.0 pM.

The results of two experiments using alternate substrates are shown in Figs. 4 and 5. When NAD+ and NADP+ were present, each at a concentration approximately equal to its K,, and glucose 6-phosphate was varied, a curved reciprocal plot was obtained (Fig. 4). This experiment has been repeated several

I I 4.0 8.0 Ii.0

I/tNAD+I I/mM

FIG. 3. Double reciprocal plot showing alternate product inhibition by NADPH of NAD-linked reaction at saturating concentration of glucose B-phosphate (3.3 mM) and various concentrations of NAD+. Buffer was as in A. The concentrations of NADPH used were 1, 0; 2, 25 PM; S, 50 pM; 4, 75 PM; 5, 100 pM.

I-

/ I-

d

FIG. 4. Double reciprocal plot from alternate substrates experiment. The buffer used was as in Fig. 2A. The glucose 6-phosphate concentration was varied as indicated, and the following additions were made. 0, 14 /AM NADP+; A, 0.25 mM NADf: 0, 7.0 /.tM NADP+ plus 0.125 mM NAD+.


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Issue of April 10, 1971 C. Olive, M. E. Geroch, and H. R. Levy 2051

times under slightly different conditions and curvature was clearly evident each time. The curvature is also evident when the data are plotted in the form of s/v versus s or v versus v/s. Fig. 5 illustrates an experiment in which glucose 6-phosphate and 2-deoxyglucose 6-phosphate were present in a mixture at concentrations approximating their respective K, values and NAD+ was varied. A linear reciprocal plot was obtained.

Eject of Varying pH-Initial velocity experiments were performed in the following buffers: Tris-maleate-NaOH, pH 5.74 to 7.21; imidazole-HCl, pH 6.44 to 7.54; Tris-HCl, pH 7.52 to 9.05; and Ammediol-HCl, pH 8.46 to 9.90. Altogether 17 experiments were performed, each one comprising duplicate or triplicate (see “Experimental Procedure”) assays at five concentrations of NAD+ at each of five different concentrations of glucose B-phosphate.

Reciprocal plots and replots of intercepts versus reciprocal changing-fixed substrate concentration were prepared as described above. All reciprocal plots, except for one experiment, were linear and gave patterns which intersected to the left of the ordinate and above the abscissa. The only exception was at pH 9.90 where a suggestion of curvilinear reciprocal plots was seen when glucose B-phosphate was the varied substrate (although the data were readily fitted to a straightline function) ; with NAD+ varied at this pH, linear reciprocal plots were seen. For all experiments the replots were linear, and in all instances the values for V from the two replots were in good agreement.

The kinetic constants obtained from this series of experiments are summarized in Table II and plotted in Fig. 6 according to the procedure outlined by Dixon (21) and Dixon and Webb (22).

When these experiments were performed initially no attempt was made to control differences in ionic strengths of the various buffers. For the imidazole-HCl, Tris-HCl and Ammediol-HCl buffers, the ionic strength of each buffer varied, over the pH range employed, from approximately 0.019 at its lowest pH to 0.0037 at its highest pH. For the Tris-maleate-NaOH buffers the ionic strengths were much higher. It was assumed that V was largely unaffected by changes in ionic strength above pH 7 since log V remained constant between pH 7 and 10. This would not be true if the calculated differences in ionic strength pro- duced substantial effects on V.

The experiments in Tris-maleate-NaOH buffers were repeated, maintaining the ionic strength at 0.018 to 0.019 with NaCl. It is the data from these experiments which are shown in Fig. 6. For each of the other three buffers, appropriate control experiments were conducted at the highest pH employed and with the ionic strength adjusted to 0.019 with NaC1. These experiments showed that the K, of NAD+ was insensitive to ionic strength over the range tested but that the K, for glucose 6-phosphate was affected. This effect is greatest at pH 9.0 where the Km at the higher ionic strength is approximately 40y0 of that at low ionic strength (Table II). This difference produces a change of less than 10% in the value of pK, for glucose 6-phosphate. The points plotted in Fig. 6 have been corrected in the manner specified but the corrections do not, in any case, substantially affect the inflection point involved.

Effect of Sulfhydryl Inhibitors-The pH studies of the NAD- linked reaction suggested that one or more groups on the enzyme with pK’ = 8.5 to 9.0 is involved in binding glucose 6-phosphate. Both -SE1 groups and e-NH2 groups can fall in this range. DeMoss, Gunsalus, and Bard (8) found that the partially puri-

I/(NAD+l I/mM

FIG. 5. Double reciprocal plot from alternate substrates experiment. Buffer was as in Fig. 2A. The NAD+ concentration was varied as indicated, and the following additions were made. l , 24 mM 2-deoxyglucose 6-phosphate; A, 0.12 mM glucose 6-phosphate; 0, 12 mM 2-deoxyglucose g-phosphate + 0.06 mM glucose B-phosphate.

TABLE II

Kinetic constants at various pH values

pH of buffer V’L ;lucose-6-I Km WAD+ K, 1 1 \iAD+ Ki

--__ AA/&n IrM PM PM

Tris-maleate-NaOH 5.74 .................... 6.27 .................... 6.77 .................... 7.21....................

Imidazole-HCl 6.44 .................... 6.63 .................... 7.21.................... 7.54 .................... 7.54 ....................

Tris-HCl 7.52 .................... 7.80 .................... 8.43 .................... 9.05 ....................

9.05 ....................

Ammediol-HCI 8.46 .................... 8.90 ....................

9.17 .................... 9.57 .................... 9.90 .................... 9.90 ....................

0.0467 76.6 548 934

0.0707 68.2 432 1250 0.0996 66.4 359 950

0.110 74.1 212 839

-

-_

I

I

I

I

I

I

-

0.0920 56.8 330 978

0.104 54.9 290 1010 0.116 70.7 209 784 0.118 116 140 917

77.0b

0.110 56.2 158 731 0.0890 52.7 106 763 0.0980 79.8 77.7 1030 0.0890 153 80.0 1950

62.0b

0.105 62.9 121 973 0.108 96.2 114 906 0.112 190 133 923 D.106 238 73.2 2440 0.109 877 38.4 1760

5506 i

0 Average of V from both reciprocal plots, standardized as ex- plained in text.

b Value corrected for ionic strength (see text).


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2052 Kinetics of Glucose 6-Phosphate Dehydrogenase Vol. 246, No. 7

-1-55.0 I I I I I

6.0 7.0 8.0 9.0 10.0

PH

60-

! E

FIG. 6. The effect of pH on kinetic constants of the NAD-linked reaction. l , Tris-maleate-NaOH; 0, imidazole-HCI; W, Trio- HCI; 0, Rmmediol-HCl. The compositions of the ordinates, in terms of rate constants, are, for A,

for B,

for c,

log (2%;) ;

log(EJ;

for D,

log

and for E,

log (k-v%).

No distinction has been made between different buffers in drawing the solid lines (---) through the experimental points. The dashed lines (- - -) are drawn with slope = $1, 0, or -1, and the intersection points are 0.3 log units above or below the data lines, as described by Dixon (21, 22).

fied glucose B-phosphate dehydrogenase from L. mesenteroides was inhibited by iodoacetate. We tested the effects of iodoacetate, iodoacetamide, and p-hydroxymercuribenzoate on both the N$DP-linked and the NAD-linked reactions. En-

I I / I I 0.1 0.2 0.3 04 0.5

PYRIDOXAL PHOSPHATE mM

FIG. 7. The effect of pyridoxal 5’-phosphate on glucose B-phosphate dehydrogenase activity at two different pH values. En- zyme was incubated at room temperature in the dark in 0.03 M

potassium phosphate buffer, pH 6.32 or pH 7.66, with the given concentration of pyridoxal 5’-phosphate. At 15 min, 0.01 ml was removed and added to a reaction mixture containing 0.03 M potassium phosphate, pH 7.66, 1.0 mM NAD+? 0.5 mM glucose 6-phosphate, and pyridoxal 5’-phosphate of the same concentration as that used in the incubation.

zyme (10e8 to lo-lo M) was incubated for 30 min at 25” in buffer (Tris-chloride, pH 7.8 or pH 9.01, buffer + 0.083 IIIM

NADP+, buffer + 2.5 InM NAD+, or buffer + 3.3 mM glucose B-phosphate, with each inhibitor at a concentration of 1 .O InM,

0.1 mM or 0.01 mM. No inhibition was noted of either the NADP-linked or NAD-linked reaction under any of these experimental conditions.

Effect of Pyridoxal 5’-Phosphate-Pyridoxal 5’.phosphate has been utilized to detect the e-amino group of lysine at the active site of various enzymes, including glucose B-phosphate dehydrogenase from C. utilis (23). Pyridoxal 5’-phosphate was found to inhibit glucose B-phosphate dehydrogenase from L. mesenteroides. Fig. 7 illustrates the concentration dependence of pyridoxal 5’-phosphate inhibition in phosphate buffers at at pH 7.66 and pH 6.32. Under the conditions of this experi ment, 0.1 mM pyridoxal 5’-phosphate produces 50% inhibition at pH 7.66; at the lower pH more pyridoxal 5’-phosphate is required to give the same degree of inhibition. Table III demonstrates the fact that the enzyme must be incubated with pyridoxal 5’-phosphate prior to assaying in order to achieve maximum inhibition. It can also be seen in Table III that if pyridoxal 5’-phosphate is omitted from the assay mixture, the inhibition is progressively diminished, demonstrating that the inhibition is reversible. Finally, pyridoxamine 5’phosphate and pyridoxal are only slightly inhibitory, demonstrating spcci- ficity for the inhibition by pyridoxal5’-phosphate.

The kinetics of inhibition by pyridoxal 5’-phosphate were


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examined. When glucose B-phosphate was varied at a constant, nearly saturating concentration of h’ADf, pyridoxal 5’-phosphate behaved like a competitive inhibitor (Fig. 8A) with Ki = 0.039 mM. When NAD+ was varied and glucose B-phosphate maintained at nearly saturating concentration, inhibition by pyridoxal 5’-phosphate was noncompetitive (Fig. 8B). Although, in these experiments, the varied substrate was in- cluded in the 15-min incubation of the enzyme as well as in the

TABLE III

Ej’ects of pyridoxal 5’-phosphate and analogues

I<nzyme was incubated at 25” in the dark in 0.03 M potassilun phosphate buffer, pH 7.66, with the additions noted in the table under “Incltbation.” An aliyuot, of this mixture was removed at 15 min, added to a cuvette contailliug NSD+ (1.0 rn~~), ghlcose 6-phospha1.e (0.5 IIlM), and the additions noted Imder “Assay” in 0.03 M potassium phosphate buffer, and the reaction rate measured for 5 min. The rates given are those measured 15 set and 4 min, respectively, after the assay was initiated. In Experiment 4 the assay mixture contained no pyridoxal 5’.phosphate; the concentration given is due to dilution from the incnbation mixture.

Experi- ment

Additions”

Incubation

None PT,P, 0.5 Inn% None PLP, 0.5 rnM PMP, 0.5 rnM Pyridoxal, 0.5 mu

- Assay

None PT,P, 0.5 mM PLP, 0.5 rnM

PLP, 5.0 pL-“I PMP, 0.5 rnhf Pyridoxal, 0.5 nl~

I-

Rateb

15 set 1. 100

25 89 25 81 92

4 min

100 25 44 47

u PLP, pyridoxal 5’.phosphnt,e; PMP, pyridoxnminc 5’.phosphate.

b The rates are given as a percentage of the rates in Experiment 1.

I t I I A

0 0.5 1.0 1.5 2.0

I/(G6P) I/mM

assay, separate experiments demonstrated that the same degree of inhibition was obtained with 0.1 mM pyridoxal 5’-phosphate regardless of the presence or absence of either substrate during the incubation. The results of these studies with pyridoxal 5’-phosphate are consistent with the involvement of an c-NH2 group of lysine in binding of glucose 6-phosphate to the enzyme.

DISCUSSION

Kinetic Mechanisms for NADP-linked and NAD-linked Reactions-Soldin and Balinsky (24) investigated the kinetic mechanism of glucose 6-phosphate dehydrogenase from human erythrocytes. They showed that the mechanism of this reaction (NADP-linked) is sequential, but they were unable, from their experiments, to rule out a Theorell-Chance mechanism or a rapid equilibrium random mechanism in which NADPH acts also as a dead-end inhibitor combining with the enzyme glucose B-phosphate complex. The data of Sanwal (3) on glucose 6-phosphate dehydrogenase from B. coli a,re compatible with an ordered sequential mechanism with NADP+ combining with free enzyme and NADPH being the last product released, but these studies also do not rule out other mechanisms.

Initial velocity studies on the NADP-linked reaction of glucose 6-phosphate dehydrogenase from I,. mesenferoides are consistent with a sequential mechanism. Product inhibition studies, using NADPH, are consistent with an ordered mechanism with the oxidized coenzyme being bound first and the reduced coenzyme released last. Further support for this

mechanism is provided by the experiment using a mixture of NM)+ and NAI>P+ with glucose 6-phosphate (Fig. 4). A rapid equilibrium random mechanism is ruled out by this experiment but a Theorell-Chance mechanism is not excluded since no information is provided by these experiments concerning the existence of significant steady state levels of the ternary complex.

The data for the NAD-linked reaction point to a more com-

50-

40-

t 2

30-

FIG. 8. 8, double reciprocal plot showing competitive inhibi- centrations used were 0,O mM; l ,0.05 mM; q ,O.l mM. B, double tion of the NAD-linked reaction by pyridoxal 5’-phosphate with reciprocal plot showing noncompetitive inhibition of the NAD- respect to glucose B-phosphate. The enzyme was incubated for linked reaction by pyridoxal5’-phosphate with respect to NAD+. 15 min at room temperature in the dark in 0.03 M potassium phos- The experiments were conducted as described for A except that phate, pH 7.66, and the indicated concentrations of glucose 6-phos- the indicated concentrations of NAD+ were present during the phate and pyridoxal 5’-phosphate. The reaction was then initi- incubation and the reaction was initiated by adding 4.0 mM glucose ated by adding 1.0 mM NAD+. The pyridoxal 5’-phosphate con- B-phosphate. Symbols are as for A.


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2054 Kinetics of Glucose B-Phosphate Dehydrogenase Vol. 246, No. 7

plex mechanism for this reaction. The mechanism is clearly sequential and the alternate substrate experiments (Figs. 4 and 5) establish that the addition of NAD+ to the enzyme precedes combination with glucose 6-phosphate. Thus the addition of substrates is ordered and, again, the rapid equilibrium random mechanism can be excluded. However, the patterns resulting from inhibition using the product, NADH, or the alternate product, NADPH, demonstrate that the second half of the mechanism can not be simply the ordered release of products from the same form of the enzyme, no matter which product is assumed to be released first. NADH and NADPH were both shown to be noncompetitive inhibitors with respect to NAD+ and NADH was also shown to be noncompetitive with respect to glucose 6-phosphate. The previous report of competitive inhibition by NADPH of the NAD-linked reaction (11) could not be confirmed. The earlier, preliminary experiments were performed with less pure NAD+.

The following possible mechanisms may be considered: Mech- anism a, release of products in random order; Mechanism b, release of 6-phosphogluconolactone before NADH and combination of NADH with the enzyme-NAD+ complex as well as with free enzyme; and Mechanism c, the same, ordered release of products and isomeriaation of free enzyme (the “iso ordered hi bi” mechanism of Cleland (16)).

In order to distinguish between these possibilities, it would clearly be desirable to use the other product for inhibition studies as well as to study the reverse reaction. The lability of B-phospho-6-gluconolactone (25) renders such experiments difficult except at low pH. Nevertheless, a tentative choice among the above mechanisms can be made on the basis of the data reported here. Mechanisms a and b do not account for the noncompetitive inhibition by NADPH with respect to NAD+ (Fig. 3). There is no evidence for either random release of products or of addition of NADPH at a second point in the mechanism of the NADP-linked reaction. Noncompetitive inhibition by NADPH with respect to NAD+ can not be reconciled with the evidence which demonstrates that both NAD+ and NADPII react with free enzyme, unless there are two forms of free enzyme. These considerations point to 1Mechanism c, in which NAD+ reacts with one form of the enzyme (E) and NADH and NADPH react with an isomerized form (E’). All the data fit this mechanism except for the experiment illustrated in Fig, 20. Under conditions of saturating NAD+, kinetic isolation would be imposed between the points of addition of NADH and glucose B-phosphate; the inhibition should be uncompetitive. The concentration of NAD+ used in this experiment, which has been repeated several times with the same result, was 55 K,. It is possible, of course, that at still higher concentrations of NAD+ the inhibition may become uncompetitive, but this does not seem likely. A modification of this mechanism, however, is consistent with all the data if it is assumed that added NADH can form a dead-end complex by reacting with the enzyme-NADf complex as well as with B’, the isomerized form of free enzyme. Addition of NADH at this points leads to a competitive effect with respect to glucose 6-phosphate and this effect, together with the uncompetitive effect caused by its interaction with E’, results in the observed noncompetitive inhibition. It is also possible that NADH reacts with E as well as with E’, since the observed, noncompetitive inhibition with respect to NAD+ may be the result of a combined competitive and noncompetitive effect. Although

such a combination appears possible a priori, we have no evidence for it and it is not required by the kinetic data. Our data also do not rule out a Theorell-Chance mechanism for the NAD- linked reaction for the same reasons as those cited for the NADP- linked reaction above.

The mechanism proposed for the two reactions catalyzed by L. mesenteroides glucose 6-phosphate dehydrogenase is illustrated in Fig. 9. Isomerization of free enzyme has also been proposed to occur in the mechanism of the reaction catalyzed by NADP- linked malic enzyme in E. coli (26).

The initial rate equation for the NADP-linked reaction is given in Equation 3, in which A’, B, P, and Q’ represent

k’+,k’+zk’+,k’+4A’BE’o ’ = k’-,k’+e(k’-2 + k’+g) + k’+~k’+&‘-2 + k’+dA’ (3)

+ k’+zk’+ak’+a + k’+~k’+dk’+a + k’+&4’B

NADP+, glucose g-phosphate, 6-phospho-Sgluconolactone, and NADPH, respectively, and the appropriate rate constants are designated with a k’, as in Fig. 9, to distinguish them from those for the NAD-linked reaction. The composition of various kinetic constants is given in Table IV. The initial rate equation for the NAD-linked reaction is given in Equation 4, in which A, B, P, and Q represent NADf, glucose B-phosphate, 6-phospho-6-gluconolactone, and NADPH, respectively.

k+,k+zk+ak+&+sAB.%

u = k--lk+&k + k+J (k+s + k-5) + k+lk+rk+r(k+z + k+3L4 __-

+ k+&+&,dk+~ + MB + k+&+dk+&+4 (4)

+ k+&+~ + k+dc+sM B

It will be noted that both Equations 3 and 4 are in the form of Equation 2, but that the collections of rate constants which comprise the kinetic constants differ.

Equations 3 and 4 may be rearranged in various ways to facil- itate expression of the equations for reciprocal initial velocity plots and replots in terms of rate constants, as has been done by Volini and Westley (27). For example, in experiments on the NAD-linked reaction in which glucose 6-phosphate is the varied substrate, Equation 4 is rewritten as follows

The terms inside the first brackets represent the slope of the reciprocal initial velocity plot and the terms inside the second brackets represent the ordinate intercept. Each of these collections of terms is itself in slope-intercept form and gives the equations for the replots. Thus, when the slopes are plotted versus reciprocal NAD+ concentration, the slopes and intercepts of this replot are obtained, respectively, from the first and second terms inside the brackets of Equation 6

Slope = i [k-l ($$$)(“z?$+“-“)] i

-i-&y

(6)

When intercepts from the reciprocal initial velocity plot are plotted versus reciprocal NAD+ concentration, the slopes and


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Issue of April 10, 1971 C. Olive, M. E. Geroch, and H. R. Levy 20.53

(E,NAD+) (E! NADH) (E!NADPH) (E’NADP+)

66p~(~.~.).,r,6p6~r:,(~.~.)~...

i +2

6PGA

FIG. 9. Kinetic mechanisms proposed for the NAD- and NADP- linked reactions catalyzed by glucose g-phosphate dehydrogenase. The formulation used is that of Westley (14). Enzyme complexes are given in parentheses. T.C., the ternary complexes; G6P. glucose B-phosphate; GPGL, 6-phospho-&gluconolactone; GPGA,

intercepts of this replot are obtained from the first and second terms inside the brackets of Equation 7

Intercept=&[e]$+j$;+&+&] (7)

These equations are useful because they contain the relevant kinetic constants in the form of rate constants. Thus the K, for NADf is obtained by dividing the first term in brackets of Equation 7 by the second term in brackets of the same equation. The latter term is the reciprocal of the maximum velocity (in the forward direction). K, for glucose 6-phosphate is given by dividing the second term of Equation 6 by the second term of Equation 7 and Ki for NADf is obtained by dividing the first term in brackets of Equation 6 by the second term in brackets of the same equation.

For purposes of comparison the composition of the various kinetic constants is given in Table IV. The isomerization step in the mechanism for the NAD-linked reaction results in more complex kinetic constants than in the mechanism for the NADP- linked reaction. For example, the dissociation constant of the enzyme-NADf complex is not equal to KiA and is, therefore, not given by the abscissa coordinate of the intersection point of the appropriate primary reciprocal plot. In fact, this dissociation constant can not be calculated from the data obtained

in this study. By contrast, the dissociation constant of the NADP+-enzyme complex is equal to KiA for this reaction and is readily obtained from Fig. 1A.

The isomerization of enzyme may be important for the normal functioning of glucose B-phosphate dehydrogenase in L. mesenteroides. Kemp and Rose (28) demonstrated that the NAD- linked and NADP-linked reactions catalyzed by this enzyme serve different functions in viva. The NADPH generated at this step is used for reductive biosynthesis, particularly of fatty acids, whereas NADH is required for the energy-yielding formation of the fermentation products. Glucose 6-phosphate dehydrogenase thus catalyzes the formation of two alternate products which possess distinctly different metabolic functions under, presumably, different circumstances. No other major

6-phosphogluconic acid. The two postulated isomeric forms of the enzyme are designated E and E’. Rate constants for the NAD-linked reaction are marked k; those for the corresponding steps of the NADP-linked reaction are marked k’.

COP stanta

Kn

KS

v

KiA

TABLE IV

Composition of kinetic constants

NAD-linked reaction NADP-linked reaction

- -.___

k,a + k-5 k+&+& k’, sk’+n

k+&+s c k+3k+4 + k+sk+b $ k+dk+s ) k’+l(k’+a + lc;4)

k-2 + k+z

c

k+ak+&+s k’+d(k’--2 + k’,r,)

k+&+a kt&+ + k+akti, + b&+5 k’t&‘t3 + k’+J

k+dc+Jc+sEo k’+3kf+&

kt& + L&s + h&s k'ts + k’+,

k-l(k+s + k-a) Id-1

k+&t~ k't,

5 IcA = Km for NAD+ or NADP+; Kg = K,,, for glucose 6-phosphate; V = maximum velocity in forward reaction; Kin = an inhibitor constant for NAD+ or NADP+ (cf. Cleland (1G)). For location of rate constants see mechanism in Fig. 9.

source for NADPH generation has been found in this micro- organism. Conceivably, the regulation of these two reactions catalyzed by the same enzyme is most effectively achieved by providing two different forms of the enzyme for combination with NADf and NADPf and, possibly, by providing for a mechanism to regulate their interconversion. In this connection, evidence has been provided that rat mammary gland glucose 6-phosphate dehydrogenase reacts with NAD+ as well as with NADPf (5) and that two different forms of that enzyme, presumably conformational isomers, react with the two coenzymes (6, 7). In the case of the mammary enzyme, various reagents and conditions have different effects on these two activities and this difference has been attributed to their effect on the equilibrium between the two enzyme forms (6, 7). Whether a similar mechanism operates to regulate the two reactions catalyzed by glucose 6-phosphate dehydrogenase in L. mesenteroides

is not yet known, but those reagents and conditions which affect the two reactions of the mammary enzyme differentially


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2056 Kinetics of Glucose 6-Phosphate Dehydrogenase Vol. 246, n:o. 7

do not exert differential effects on the NAD-linked and NADP- linked activities of the bacterial enzyme (11).

EJects of pH, --XH Reagents, and Pyricloxal 5’-Phosphate- Some caution is required in interpreting the results of the pH studies. The meclranism for the NAD-linked reaction was deduced from experiments conducted at pH 7.8. An implicit assumption in the detailed interpretation of the pI1 studies is that exactly the same mechanism holds over the entire pH range employed (i.e. including the isomerization) . Further- more, the kinetic mechanism is suliiciently complex that variations of kinetic constants with p1-I may not reflect variations of rate constants associated with a single step of the mechanism. For example, the term log F/K, contains rate constants for two steps in the mechanism (see legend to Fig. 6): the reversible association of glucose 6-phosphate with the binary enzyme- NM)+ complex and the dissociation of 6-phospho-&gluconolactone from the ternary complex. Without further experiments it can not be determined which step is rate limiting or whether one step may dominate at one pH and the other step at a different pII. We have assumed, however, that variations in log V/K, with $1 reflect changes in k+z/kwz. Finally, as is well known, the ionization of a group in an enzyme may be influenced dras- tically by its microenvironment. Despite these limitations, the

elucidation of the effects of p1-I changes on the kinetic constants of a reaction can provide useful guides for further experiments and in the present study it has led to the tentative identification of an amino acid involved in binding glucose 6-phosphate.

Fig. 6A shows that V is unaltered between pH 6.5 and pI1 9.9; below pH 6.5 there is a small decrease in log V, insufhcient to permit assigning of any pK’ value. Thus no conclusions can be reached from these studies concerning the groups on the enzyme which participate in catalysis. However, it is not known

which of the three rate constants comprising V (Table IV) defines the rate-limiting step in the mechanism and the insensitiv- ity of V to pH does not ensure that each of the three unimolec- ular steps (in the forward direction) is unaffected. Further experiments are required to resolve this question.

Plots of pKb and pK, versus pJ-I are not shown because they are virtually identical with the plots of log V/KB and log V/Ka versus pII, respectively, and because the latter terms are less complex quotients of rate constants. The data in Fig. 6B pro-

vide clear evidence that a group on the enzyme with a pK’ of approximately 8.9 is involved in glucose B-phosphate binding. The slight curvature below PIT 6.5 in this curve may be a result of the same effect seen in Fig. 6A. If so, both must involve the dissociation of 6-phospho-&gluconolactone from the ternary complex, since Jc+~ is the only rate constant shared by V and V/K,. A more likely cause for the curvature in Fig. 6B is the ionization of the phosphate group of glucose 6-phosphate, which has a pK’ of 6.1 (29) and which should exist in the anionic form in order to bind to the protonuted E-NH* group of lysine.

The curves in Fig. 6, C, D, and E, all relate to changes in rate constants concerned with the reversible interaction of enzyme with NAD+ and with enzyme isomerization. In Fig. 6, D and B, the experimental points at high pH do not appear to fit well to the curves. This is probably because the values for Kia (which were calculated from the experimental data using Equa- tion 2) could not be corrected for ionic strength variations. The point at pH 9.05 is most seriously affected (see Table II) and it has been omitted in fitting the curves in these two figures.

Despite these uncertainties, the main features of Fig. 6, C, D, and E, stand out clearly. Two groups on the enzyme are involved in the dissociation of NAlY+ from the binary complex (Fig. 6E) and probably the same groups are concerned either with the binding of NAD+ to enzyme or with the isomerization step (Fig. 6C). The close similarity between these two curves accounts for the fact that KiA is virtually insensitive to pIT over the range tested, although there may be some effect above pH 9. The two groups on the enzyme have pK’ values of 6.7 to 7.0 and 9.0 to 9.5. This suggests that the first group is the imidazole of histidine and the second group a sulfhydryl of cysteine, an e-amino of lysine, or a hydroxyl of tyrosine. We have found no evidence for the involvement of either an -SIT group or an E-KHZ group in NADf binding. Both histidine (30) and tyrosine (31) have been implicated in the mechanism of action of glucose B-phosphate dehydrogenase from C. utilis. Further experiments are required to identify these groups in the L. mesenteroides enzyme and to provide evidence which will permit one to assign their role to either NAD+-enzyme interaction or enzyme isomerization.

Probably the most clear-cut result of the pI1 studies is the inflection at pH 8.9 in Fig. 6B. We therefore attempted to obtain further evidence for the identity of a group on the enzyme with a pK’ near 9 which could be involved in binding glucose B-phosphate. The most likely candidate appeared to be an -SII group of cysteine since DeMoss et al. (8) had reported substantial inhibition of crude glucose 6-phosphate dehydrogenase from L. mesenteroides by 15 KM iodoacetate. Our failure to demonstrate any inhibitory effect of iodoacetate, iodoncetamide, or p-hydroxymercuribenzoate, even under conditions of pro- longed incubation with high concentrations of these reagents, does not confirm the results of DeMoss et al. but is consistent with similar data reported for glucose 6phosphate dehydrogenase from C. utilis (32).

Suggestive evidence was obtained, however, for the participation of the e-NH1 group of a lysine residue in binding glucose 6-phosphate. Pyridoxal 5’-phosphate gave reversible, specific, pII-dependent inhibition of the enzyme which was noncompetitive with respect to NAD+ but competitive with respect to glucose B-phosphate. The fact that the extent of inhibition was not altered by the inclusion of NAD+ or glucose 6-phosphate in the incubation which precedes the assay is consistent with the ordered, sequential binding to the enzyme of NADf and glucose B-phosphate, required by the kinetic mechanism. The involvement of the e-NH2 group of a lysine is also in accord with the observed sensitivity to high ionic strength of glucose 6-phosphate binding, which is expected for the interaction of an anionic substrate and a cationic group on the enzyme.

Aclcnowledgments-We thank nIrs. Kathy Bleyle for excellent technical assistance. We are particularly indebted to Dr. John Westley for invaluable discussions concerning the kinetic mechanism.

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Charles Olive, Mary E. Geroch and H. Richard LevySTUDIES

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1971, 246:2047-2057.J. Biol. Chem.

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