THE JOURNAL OF Bromorca~ Crrmmm~ Vol. 249, No. 10, Issue of May 25, pp. 3132-3139, 1974

Printed in U.S.A.

Purification and Kinetic Characterization

Cation-activated Glycerol Dehydrogenase

from Aerobacter aerogenes*

of a Monovalent

(Received for publication, October 4, 1973)

W. GLENN MCGREGOR,$ JAMES PHILLIPS, AND CLARENCE H. SUELTER~

From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48823

SUMMARY

Glycerol dehydrogenase from Aerobacfer aerogenes has been partially purified. Kinetic constants for the substrates are reported and an Ordered Bi Bi mechanism based on product inhibition studies is presented. An investigation of the kinetics of monovalent cation activation shows that sub- strate affinities, particularly those for glycerol and dihydroxy- acetone, are affected by monovalent cations, but that the kinetic mechanism remains unaffected. Specifically, at pH 7.5, the affinity for NAD was not affected when assayed in 0.1 M NH&l instead of 0.1 M (CH3CH2)4NCI; the affinity for NADH was increased Z-fold. More striking was the 22-fold decrease in the K,,, value for glycerol and the 25-fold decrease in the K, value for dihydroxyacetone. The maximum ve- locity in 0.1 M NH&l was nearly 3 times that in 0.1 M (CH3CH2)4NCl with all other conditions identical. The rela- tive activating efficiency of the various cations as measured by the resultant maximum velocity, NH,+ > Tl+ > K+ > Rb+, is the same for both oxidation and reduction and is independent of pH. In contrast, the affinities for the mono- valent cation were dependent on pH. When the pH varied from 6.1 to 7.5 to 9.0, the affinity for NH.,+ and Tl+ decreased whereas the affinity for K+ and Rb+ increased. The require- ment for a monovalent cation was not eliminated by a proton.

Glycerol dehydrogenase (glycerol:NAD+ oxidoreductase, EC 1.1.1.6) is a pyridine nucleotide-linked enzyme catalyzing reaction (1).

NAD+ + glycerol e dihydroxyacetone + NADH + H+ (1)

Enzymes which catalyze the oxidation of glycerol to di- hydroxyacetone have been found in a wide variety of bacterial

* This work was supported in part by Grant GB-25119, National Science Foundation, and the Michigan State Agricultural Experi- ment Station, Michigan State Journal No. 5933.

$ Trainee of the New Jersey Rehabilitation Commission, 750 Hamburg Turnpike, Pompton Lakes, New Jersey 07442. This work constitutes partial fulfillment of the requirements of the Bachelor of Science degree in Biochemistry.

5 Recipient of Research Career Development Award 1 K3 GM 9725 of the National Institutes of Health.

systems (1, 2). Mammalian systems with glycerol dehy- drogenating activity include rat liver (3) and rabbit skeletal muscle (4) ; these enzymes have substantially different properties than those found in bacteria.

The partial purification of an NAD-dependent glycerol dehydrogenase from Aerobacter acrogenes was first reported by Burton and Kaplan (5). Lin and Magasanik (6, 7) later de- scribed the enzyme from a capsulated strain of the same or- ganism. This strain can utilize glycerol as a sole source of carbon and energy, in which case a glycerol dehydrogenase is induced. The level of enzyme in aiuo is affected by the oxygen tension during growth, with the induction of glycerol dehy- drogenase being favored under anaerobic conditions. During the course of their investigation, Lin and Magasanik found that certain monovalent cations are required for maximal catalytic activity.

Since the activity of many monovalent cation-activated en- zymes is nearly 0 in the absence of monovalent cations, the effect of the cation on the various kinetic parameters of the reaction can not be discerned. In this respect then, the study of glycerol dehydrogenase offers an opportunity since the enzyme in the absence of monovalent cation expresses 25 to 30% of that observed in the presence of the saturating ammonium cation. As a consequence of our interest in the role of monovalent cations in the activation of enzymes, we describe in this paper the partial purification of glycerol dehydrogenase from ,4. aerogenes strain 1033 and the determination of the various ki- netic parameters and rate constants for each of its substrates in the presence and absence of activating cation.

MATERIALS AND METHODS

Dihydroxyacetone, NADH, MES,’ TES, TAPS, and piperidine were obtained from Sigma. ‘Grade’V NAD, also obta’ined from Sigma. was used for the kinetic studies: otherwise erade III NAD

Y I

(Sigma) was used for routine assays. Spectroquality glycerol from Matheson, Coleman, and Bell was used in the kinetic studies; analytical grade glycerol from Mallinckrodt was used in all other cases. Tetraethylammonium chloride was purchased from East- man Organic Chemicals and recrystallized from absolute ethanol. Tetraethylammonium hydroxide was obtained by passing a 2.0 M solution of (CH&Ht)4NCl over a Dowex l-X8 anion exchange column in the hydroxyl form. Sephadex G-25 and G-200 are

1 The abbreviations used are: MES, 2-(N-morpholino)ethane- sulfonic acid; TES, N-tris(hydroxymethyl)methylS-aminopro- panesulfonic acid; TAPS, tris(hydroxymethyl)methylaminopro- panesulfonic acid.

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products of Pharmacia Fine Chemicals and DEAE-cellulose was purchased from Sigma. Ammonium sulfate was Mann enzyme grade; all other chemicals were reagent grade. Reagents for analytical polyacrylamide gel electrophoresis were Canalco prod- ucts. Glass-distilled water was passed over a mixed bed ion ex- change resin before use.

After fines were removed, DEAE-cellulose was washed succes- sively five times with 0.1 N NaOH, five times with 0.1 N HCl, and finally with deionized water until the eluate was neutral. The resin was suspended in 2.0 M glycerol, 0.1 M KCl, and 0.05 M (CHs- CHz)hN+-TES, pH 7.5. Sephadex G-200 was defined and allowed to swell for 3 days in a solution of 2.0 M glycerol, 0.1 M KCl, and 0.05~ (CH&Hs)4N+-TES, pH 7.5. After degassing, the resin was poured into a 2.5-cm diameter reverse flow column and gravity packed to a height of 70 cm. Solvent was passed through the col- umn in the downward direction for several hours to pack the resin. Chromat,ography was accomplished by reverse flow. The void volume and the internal volume were determined by using a mix- ture of 0.2% blue dextran and 1 mM cysteine vinyl quinoline (X,,, 310 nm). The column was used and stored at O-4”, and was re- equilibrated with 0.1 N KC1 and 0.05 M K+-cacodylate to prevent microbial growth when not in use.

Protein concentrations were determined by the method of War- burg and Christian (8). Routine assays of enzyme activity were made by adding an appropriate aliquot of solution (typically, 10 to 25 ~1) to the reaction mixture in a lo-mm light oath l-ml quartz cuvette at 28”. The increase in optical density at 340 nm was recorded with a Gilford model 220 modified Beckman DU suectro- photometer equipped with a Sargent model SRL recorder. The reaction mixture contained 10 rnk NAD, 0.10 M glycerol, 0.10 M

NH&l. and 0.05 M (CHXH.Z)~N+-TAPS. DH 9.0. The initial rate - , ~ - - I - I 1

is converted to micromoles of NADH formed per min per mg of protein by dividing the change in optical density by the final protein concentration times 6.22 (9).

The enzyme solution used in the enzyme kinetic experiments was desalted on Sephadex G-25 equilibrated with 0.1 M (CHS- CHz)dNCl. The desalting was completed immediately before the kinetic assays, since the enzyme lost activity in the absence of glycerol. Before use, sodium-NADH was passed over Sephadex G-10 equilibrated with (CH&HZ)~NCI to replace the sodium cat- ion with (CH&Ha)4N+. Unless otherwise stated. saturating sub- . .~ strate concentr,ations refer to 100 mM glycerol, 100 mM dihydroxy- acetone, 10 mM NAD, and 1 mM NADH. When monovalent cat- ions were varied in the presence of saturating substrates, the ionic strength was controlled at 0.1 M with (CH&H2)4NCl. The rea- gents used for determinations in the absence of ammonia were tested for trace NH4 by the method of Livitski (10) ; in no case was significant contamination found.

Results of the purification are summarized in Table I. Purity 0s Enzyme-The protein obtained by the preparation

described above is not homogeneous as several bands were ob- served when polyacrylamide gels were stained with Coomassie

blue after electrophoresis. However, the catalytic activity corresponded with the major protein band; a faint minor activity band estimated at less than 5y0 of the total activity was also present. Similar chromatograms were obtained at several points in the activity peak of the elution profile.

Polyacrylamide gel electrophoresis was performed as suggested by Mauer (11) in 6.0% gel at 25”. The samples were applied in 5Ooj, (v/v) glycerol in Tris-phosphate pH 6.9 and were elect,ro- phoresed at 5 ma constant current per tube in standard Tris- glycine buffer at pH 8.3. The gels were stained for protein with Coomassie blue, and the activity was determined by placing the gels in an assay medium containing 10 mM glycerol, 10 mM NAD, 100 mM NH4+, nitroblue tetrazolium, N-methylphenazonium methosulfate, and 50 mrvr (CH$Hg)4N+-TAPS, pH 9.0.

A. aerogenes strain 1033 was a gift from Dr. Edmund C. C. Lin. The bacteria were maintained by periodic t,ransfers on peptone bovine extract agar slants.

pH Opti?rlu?n-Specific activity as a function of pH is shown in Fig. 1 for the osidation of glycerol and in Fig. 2 for the re- duction of dihydroxyacetone. If it is assumed that tetra- ethylammonium ion functions only as an ionic strength effector, then the relative effects of the three cations are immediately ap- parent; ammonia is, in general, a much more effective activator. Further, the requirement for a monovalent cation cannot be substituted by H+ at the lower pH values. It is clear that the

The cells were sonically disrupted by treatment for 10 to 15

min in a Raytheon model DF 101 250-watt sonic oscillator. This preparation was centrifuged at 50,000 X g for 20 min to yield Fraction I. One per cent protamine sulfate (neutralized with (CH3CH2)4NOH) was added dropwise with stirring at 4”

to a final concentration of 0.06 mg per mg of protein. The supernatant after centrifugation contains the enzyme activity and is designated Fraction II.

Solid ammonium sulfate (14.4 g per 100 ml) was gradually

added to Fraction II to give 25$& saturation. The precipitate was removed by centrifugation and the supernatant (Fraction

III) was brought to 55% saturation by the slow addition of

solid ammonium sulfate (20.7 g per 100 ml). The precipitate was collected and dissolved in a minimum volume of 2.0 M

glycerol, 0.1 M KCl, 2 mg of phenylmethylsulfonyl fluoride per liter, 0.05 M (CH&HZ),N+-TES, pH 7.5, yielding Fraction IV. During the (NH&SO4 precipitation the pH was maintained at 7.5 by the periodic addition of 1% (CH,CH2)4NOH.

Fraction IV was applied to the reverse flow Sephadex G-200

column described in “Materials and Methods” and eluted with

2.0 M glycerol, 0.1 M KCl, and 0.05 M (CH&Hz).,Nf-TES, pH 7.5. Fractions of 5 ml were collected, and activity was found between 140 to 440 ml. Those tubes with the highest specific activity were pooled (Fraction V) and applied to a DEAE- cellulose column. The activity was eluted at approximately 0.25 M KC1 when a linear concentration gradient was applied

ranging from 0.1 M to 0.5 M. The fractions containing the

highest activity were pooled to give Fraction VI.

RESULTS

TABLE I

Purifccation of glycerol dehydrogenase I

Fraction Total Specific activitya Yield ‘urified

PuriJcation-The enzyme was prepared from A. aerogenes ml m&- ,m,,* -fold

strain 1033. The basal growth medium was the same as that I. Cell lysate. 75 4780 3346 0.7 00 1 described by Lin and Magasanik (6), with the exception that II. Protamine sul-

guanine and glucose were not included. One liter of the sterilized fate.... 85 2650 2915 1.1 87 1.5

medium was aseptically inoculated with cells from a fresh agar III. Supernatant, 25%

slant; this culture was grown overnight at 37” and was used to (NHdaSO4 100 1050 2625 2.5 78.5 3.5

inoculate a 15-liter medium. This large culture was mag- IV. Precipitate, 55%

netically stirred while growing for 24 hours at 37”. The cells (NH&SO* 12 500 2200 4.4 65.8 6.2

were collected in a Sharples continous flow centrifuge, washed V. Sephadex G-200 30 140 1750 12.5 52.3 17.8

once with distilled water, and suspended in 0.05 M phosphate, VI. DEAE-cellulose.. 25 37.5 1312 35.0 39.2 50.0

pH 7.5, containing 25 mg of phenylmethylsulfonyl fluoride per a Defined as micromoles of NADH min-1 mg-1 as determined 100 ml. All successive steps were performed at 4”. under the conditions specified in “Materials and Methods.”

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I I I I

5 6 7 8 9 IO II

PH PH

oxidation of glycerol (Fig. 1) is markedly reduced at pH 5 to 6 whereas the rate of reduction of dihydroxyacetone (Fig. 2) is essentially identical from pH 4 to 8. Since neither substrate has an ionizable group in this pH range, it is obvious that an ionizable group on the enzyme surface is either responsible for maintaining the correct enzyme configuration for proper binding of substrate or is involved in the catalysis per se. Figs. 1 and 2 also show that a similar pH dependence is observed in the presence and absence of cations, implying that the same rate- limiting step is involved in the presence and absence of cations at levels of substrates used in the assay. It should be stressed that these pH rate profiles were not obtained at saturating levels of substrate and thus the shape of the curves, particularly for glycerol oxidation (Fig. I), may not be precisely correct.

Equilibrium Constant-The reported equilibrium constant for the reaction NAD+ + glycerol * NADH + H+ + dihydroxy- acetone ranged from 5.2 X lo+ (12) to 14 X lo+ (13). In this study, the equilibrium constant was determined at a variety of hydrogen ion concentrations ranging from 5.01 X 1OV M to 5.62 x 10mg M as described in the legend to Fig. 1. The re- action mixtures were allowed to equilibrate for 2 hours at 27” before the optical densities of the solutions at 340 nm were determined and the final concentrations of reactants and prod- ucts were calculated. The results of 12 determinations gave a value of 2.38 x 10-l* moles per liter with a standard deviation of 0.58 x lo-=.

Effects of Sulfhydryl and Complexing Agents-Table II shows the relative velocities of the enzyme-catalyzed oxidation re- action in the presence of various sulfhydryl and chelating agents. The enzyme was added to the reagent in the presence of glycerol and (CH3CHz)$J+-TES, pH 7.5, and left for 12 hours at room temperature before assaying. The results show that compounds which react with sulfhydryl groups tend to inhibit the enzyme; N-ethylmaleimide, which reacts essentially irreversibly with the sulfhydryl, is a particularly powerful inhibitor. The slight activation observed with lower concentration of 2-mercapto- ethanol and dithioerythritol is consistent with the possibility that low concentrations of these compounds maintain reduced sulfhydryl groups. The inhibition by higher concentrations of mercaptoethanol or dithioerythritol may be due either to the

FIG. 1 (left). pH profile for glycerol oxidation. Buffers at 0.05 M and their pH ranges are: MES, 5.0 to 6.5, TES, 7.0 to 8.0; TAPS, 8.5 to 10.0. Buffers were titrated to the appropriate pH with tetraethylammonium hydroxide. Piperidine titrated with HCl was used at pH 11.0. All determinations were made with 10 mM NAD and 100 mM glycerol. The apparent equilibrium constant was calculated by allowing the reaction mixtures to equilibrate at 27” and then determining the con- centrations of reactants and products.

FIG. 2 (right). pH profile for dihy- droxyacetone reduction. The disap- pearance of NADH was followed by a decrease in optical density at 370 nm. NADH hm an experimentally deter- mined millimolar extinction coeffi- cient of 2.55 at this wave length. The nonenzymatic destruction of NADH was negligible down to pH 4.0. Buff - ers used were the same aa in Fig. 1, with the addition of 0.05 M (CH,- CHs)aN+-formate below pH 5.0.

TABLE II

Effecls of sulfhydryl and chelating reagents on activity of glycerol dehydrogenase

ReagenP

Control”. Water”. . EDTA.. : : :

%Quinolinol .

0-Phenanthroline.

2-Mercaptoethanold. .

Dithioerythritol .

Iodoacetamide.

N-Ethylmaleimide

Concentration Relative activity

?nM

100

5 30 53

3 65 10 79 1 116 1.85 135 0.185 100

100 0 1 110

10 1 1 116

10 1 0.1 53 0.1 0

(1 The enzyme was incubated in 0.05 M (CHaCH2)aN+-TES, 2.0 M glycerol, 0.1 M KCl, and the indicated reagent for 12 hours at room temperature.

b Enzyme in 0.05 M (CHoCHa)*N+-TES, 2.0 M glycerol, 0.1 M KCl.

0 Enzyme in distilled water with no salts or glycerol. d Inhibition by 100 mM 2-mercaptoethanol could not be reversed

by 2 m&f Zn*+, 10 mM Mn2+, or 2 mM Cop+ after removal of mercap- toethanol.

reduction of a disulfide bond or to the complexing of a metal possibly required for catalysis. That the enzyme may have a requirement for a divalent metal cation is indicated from the inhibition observed with EDTA and 8-quinolinol. The reason for the activation given by 1.85 mM 0-phenanthroline is not known, but may be due to chelation of inhibitory levels of trace metals.

Monovalent CationsThe K,, and maximum velocity attained at saturation for each activating monovalent cation are tabulated in Table III. The constants were determined using saturating

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reactions, respectively, determined in 0.1 M NH&l at the pH indicated. Figs. 5 and 6 are similar plots showing the results of determinations in 0.1 M (CH&H2)4NCl. Linear secondary plots were observed in all cases and are shown in the insets.

Product Inhibition Studies-To test the possibility that the kinetic mechanism of the reaction may be different in the

substrate concentrations, and the maximum velocities reported include the rate observed in the absence of activating cations. The activation followed Michaelis-Menten kinetics in all cases.

Examination of the results in Table III reveals several major points of interest. (a) The activation constants for K+ and Rb+ in the forward and reverse reaction at pH 7.5 are identical within experimental error. (b) A ssuming that the observations noted in Point a are true at all pH values, then the activation constants for K+ and Rb+ tend to increase as the pH is lowered. On the other hand, the activation constants of NH4+ and Tl+ tend to decrease as the pH is lowered. (c) The sequence of cation affinities at pH 6.1 (NH4+ > Tlf > K+ > Rb+) and pH 7.5 (NHd+ > K+ > Rb+) is different from that observed at pH 9.0 (NHr > Rb+ > K+ > Tl+).

Enzyme Kinetics-The initial velocity data were fitted to the Michaelis-Menten equation using a (ui)4-weighted least squares fitting routine as suggested by Wilkinson (14). Figs. 3 and 4 are primary reciprocal plots for the oxidation and reduction

TABLE III

I I

-24 -20 -16 -I2 -8 -4 0 4 8 - 24 28

FIG. 4. Initial velocity pattern for dihydroxyacetone (DHA) reduction. The kinetic assays were made with desalted enzyme at pH 6.1 in 0.1 M NH&l, 27”. The slope and intercept replota are shown in Znsets A and B.

Kinetic conslant or activating calions Sf - I pn l.Sb pH 6.1’

_-

48.0 25.4 38.1 42.6

1.25 5.50 4.32

4.26 5.26

Oxidation NH4+. . . 2.46e Rb+. . . . . 3.00 K+ . . .._... 3.27 Tl+. 4.97

Reduction NHd+...... Tl+ K+ . . . . Rb+......

55- I

n I

A- L 50 / !

.-

0.81 29.0 0.98 24.1 5.43 20.6 6.94 11.9

37.0 14.9 27.9

19.0 9.9

Q Constants were determined at pH 9.0 in 0.05 M (CH&Hz)rN+- TAPS, 27”, 25 mM NAD, and 500 mM glycerol.

b Constants were determined at pH 7.5 in 0.05 M (CH&HZ)~N+- MES, 27’, 25 mM NAD, and 500 mM glycerol.

c Constants were determined at pH 6.1 in 0.05 M (CH&Ha)rN+- MES, 27”, 10 mM.NADH, and 250 mM dihydroxyacetone.

dMaximum velocity in micromoles min-1 mg-I. 8 Corrected for NH4+ concentration.

[GLYCEROL]-’ M-’

FIG. 5. Initial velocity pattern for glycerol oxidation in the ab- sence of activating cations. The ionic strength was controlled to 0.1 M with (CH&Ha)4NCl. Conditions are otherwise the same aa in Fig. 3. ! T

;--;:: , ol, d2 d3,

-a4 -a3 -02 -aI 0 [GLYCEROL]‘mhl-’

FIG. 3. Initial velocity pattern for the glycerol oxidation reac- tion, determined in 0.1 M NH&l at pH 9.0,27”. The enzyme was desalted on Sephadex G-25 before use. The slope replot (A) and intercept replot (B) are shown in the inset. The lines are drawn according to K, and V,,, values determined statistically as de- scribed in the text.

[NADHI-‘mM-’

FIG. 6. Initial velocity pattern for dihydroxyacetone (DHA) reduction with no activators. Ionic strength was maintained at 0.1 M with (CH&Hs)4NC1. All other conditions are the same as in Fig. 4.

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presence and absence of activators, product inhibition studies were performed in 0.1 M NH&l and in 0.1 M (CH&Hz)qNC1. Figs. 7, 8, and 9 are primary reciprocal plots and Table IV~is a summary of the inhibition results.

While the data of Fig. 8 were interpreted as uncompetitive kinetics, it should be emphasized that the slope of the lines increases slightly with increasing dihydroxyacetone levels. A plot of the slope versus concentration of dihydroxyacetone is parabolic, however, the intercept replot is linear. The forma- tion of an abortive NAD-DHA complex would be consistent with these results.

I / I

5 I

2 4 6 DhM-’

FIG. 7. Product inhibition by NADH. The data were obtained by varying NAD at changing fixed levels of NADH, giving a char- acteristic competitive inhibition pattern. A replot of slopes versus inhibitor concentration showed that the inhibition is linear. The experiment was performed in 0.1 M NH&l and 1.0 M glycerol at pH 9.0, 28”.

i

FIG. 8. Product inhibition by dihydroxyacetone (DHA) in the presence of 0.1 M NH&l and 1.0 M glycerol at pH 9.0, 28”. The inhibition is linear as shown by a replot of the intercepts versus di- . .

Another alternative may be postulated if an Ordered Bi-Bi mechanism is assumed. Inspection of Mechanism 1 shows that if glycerol were not sufficiently concentrated with respect to the K,, a reversible connection would be established between the points of addition of NAD and dihydroxyacetone. Thus the inhibition would appear noncompetitive. This effect would become more pronounced in the event of a decrease in the affinity for glycerol such as occurs when NHJ+ is replaced by (CH&H2)4N+. Consequently, rather than repeat the experi- ment of Fig. 3 in 0.1 M (CH&H2)NC1, it was felt that a study of the reverse reaction would yield an equivalent amount of information. Since the K, value for dihydroxyacetone was not affected by the activator at pH 6.1, saturation by this substrate could be assured. Fig. 9 presents the results of varying NADH at changing fixed levels of glycerol with saturating dihydroxy- acetone in the absence of activators.

Kinetic Constants-The kinetic parameters for each substrate of glycerol dehydrogenase in the direction of oxidation and re- duction are given in Tables V and VI respectively. In no case was the standard deviation of the kinetic constant greater than 10% of the value of the constant; in most cases the value was significantly less than 5%. The nomenclature is that sug- gested by Cleland (15, 16), where Ka, Kb, K,, and K, indicate the Michaelis constants equivalent to the concentrations of the indicated reactants which give half of the maximum velocity when all other substrates are at infinite concentrations; Ki,, Kib, and so on, are the dissociation constants of the respective binary complexes. The kinetic constants are summarized in Table V and VI. The most salient observations here are that the kinetic constants for glycerol and dihydroxyacetone are significantly decreased in the presence of NH4+ at all pH values except for the reduction reaction at pH 6.1. The differences in the kinetic constants for NAD or NADH in the presence and absence of NHh+ were minimal, being at, most -4 times smaller in the presence of ammonia. In all cases, 0.1 M NH4+ increased the maximum velocity by 3- to 4-fold.

Since NH4+ affects Kb for glycerol, it is to be expected that glycerol would also affect the activation constant for NHd+. To ascertain the magnitude of this effect, we note that three of the constants for the reaction in Scheme 2 are known allowing

4om

/ I I I I I

-16 -12 -8 -4 0 4 8 12 16 20 24 [NADHi’mM-’

FIG. 9. Product inhibition by glycerol (GLY) without activat- ing cations. Dihydroxyacetone concentration was 1.0 M. The assavs were nerformed in 0.1 M (CH,CHl)dNC1 at nH 6.1. 28”.

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ENAD +Giy v KS

. ENAD .Gly --$ products

+ + NH: NH,+

11 K” 11 Kau (2)

ENH,+ . NAD + Gly &i% ENH: . NAD . Gly + products It is evident from the intersecting Lineweaver-Burk patterns calculation of the fourth. Specifically, Ks, KsM, and KM8 are observed in the primary reciprocal plots (Figs. 3 to 6) that the known with KM calculated from KM = [(KS. K&/K& KM binding of the substrates is sequential. Product inhibition ex- and Ks are the constants which describe the interaction of NHb+ periments performed in 0.1 M NH&l or (CH&Hz)NCl showed and glycerol with the enzyme.NAD complex. KMs is the dis- that NADH is a competitive inhibitor of NAD, that dihydroxy- sociation constant for glycerol from the enzyme. NAD .NHJ+. acetone at saturating glycerol behaves as a linear uncompetitive glycerol complex; KsM is the dissociation constant for NH*+ inhibitor with respect to NAD, and that glycerol behaves as a from the same complex. From Table V we find Ks = 44.9 mM, linear noncompetitive inhibitor with respect to NADH. Thus,

Cation

NHa+ ................ NHb+ ................ (CH&H&N+ ........

-

-

KMs = 2.07 mM, and from Table III, KsM = 1.25 mM. The cal- culated value is then KM = 74.3 mM. Thus, the affinity for NHa+ is increased nearly go-fold by glycerol.

DISCUSSION

TABLE IV Summary of product inhibition of glycerol dehydrogenase

Varied substrate Fixed sub&at@ Inhibitor Type of inhibition” KI

rnY

NAD Glycerol NADH Linear competitive 0.75” NAD Glycerol Dihydroxyacetone Linear uncompetitive 2.6” NADH Dihydroxyacetone Glycerol Linear uncompetitive 108.W

a The concentration of fixed substrates was 1.0 M.

b Inhibition constant Ki, determined from slope replot, see Fig. 7 for experimental details. c Inhibition constant Kii determined from intercept replot, see Fig. 8 for experimental details. d Inhibition constant Kit determined from intercept replot, see Fig. 9 for experimental details.

TABLE V Kinetic constants for oxidation reaction catalyzed by glycerol dehydrogenase at pH S.O$and 7.6

in presence and absence of activating moMvalent cation, NH,+

Kinetic constant“

0.1 M NHLY

rnM

K, . . . . . . 0.20 Ki,. . . . . . . . 2.42 &, . 1.25 Kd(b)c.. . . . . . . Ki.Kb/K, . . . . . . 14.5, 15.1 V,,,, pmoles min-1 mg-1. . . . . . 46

Ratio of constants, Ratio of constants.

0.1 Y (CzHs)rNCI 0.1 Y (C2Hr)4NCI 0.1 M (CzHr)rNCI 0.1 P NH&l 0.1 Y NH&I I 0.1 Y

(CzHr)rNCI 0.1 LI NH,CI

?nM ?nM m,w

0.37 1.85 0.41 0.869 2.1 2.05 0.85 5.37 5.19 0.97

32 25.6 2.07 (2.05)” 44.9 21.7 1.69 40.0 23.7

210 14.2 28 294 10.6 13.5 0.29 27 13.1 0.35

0 Kinetic constants at pH 9.0 were determined in 0.05 M TAPS buffer, at pH 7.5 in 0.05 M MES buffer. b Value obtained from an extrapolation to infinite ammonia of a plot of K,,, for glycerol obtained at various NHb+ concentrations

versus l/NHa+. c Dissociation constant for glycerol Kd(b) from the central complex was calculated from &.K,,V~/&,~~ (17).

TABLE VI Kinetic constants for reduction reaction catalyzed by glycerol dehydrogenase at pH 6.1 and 7.6

in presence and absence of activating monovalent cation, NH,+

pH 6.1 Ratio of constants, pH 1.5 Kinetic constants0 ~ 0.1 YI (CIHK),NCI

0.1 Y NH&l 0.1 M (CzHs)rNCl 0.1 YI NH&I 0.1 Y NJ&Cl 0.1 Y (GHr)rNCI

?nM mlldl

KQ . . . . . . . . . . . . . . , . 0.017 0.064 3.8 0.0054 0.010 K. ,g.. . . . . . . . . . . . . . . . 0.063 0.133 2.1 0.0095 0.024 KP . . . . . . . . . . . . . . . . . . . . . . 0.74 2.38 3.2 0.11 2.71 I&,+. . . . . . . . . . . . 0.13 2.99 KipKP/K,. . . . . 1.45, 2.7 7.10 3.4 0.19 5.80 V,,,, pmoles min-1 mg-l . . . . . 31 8 0.26 30 10.1

0 Kinetic constants at pH 6.1 and pH 7.5 were determined in 0.05 M MES buffer. b Dissociation constant for dihydroxyacetone (K,& from the central complex was calculated from KipKpVJKbVs (17).

Ratio of constants,

0.1 M (GH+NCI 0.1 Y NH&l

1.9 2.5

24.6 23 30.5 0.35

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NAD and NADH combine with the same enzyme form, and the points of addition of NAD and dihydroxyacetone along the re- action sequence are not reversibly connected when glycerol is at infinite concentration.

These results are not consistent with a Random Bi-Bi or Theorell-Chance mechanism but rather point to an Ordered Bi-Bi mechanism (17) of the form:

NAD GUY Dihydroxyacetone NADH

(E-NAD-Gly) E (E-NADH-Dihydrodyacetone)

This mechanism is also supported by the excellent agreement between the K,, calculated with the Haldane relationships de- fined for such a mechanism (16) and the value measured experi- mentally. The measured value of 2.38 x lo-l2 f 0.58 x 10-l* M is to be compared with K,, = (VI K, K,q[H+l)l(V, & Kc,) = 3.59 X lo-l2 M for reaction with NH4+ and 10.9 X lo-l2 M for reaction with (CH&H2)dNCl or K,, = (VIZ Kip K,[H+])/ (Tif K,b K,) = 4.37 X lo-l2 M for reaction with NH4f and 11.0 X lo-l2 M for reaction with (CH&H,),NCl. Since all pyridine nucleotide-linked dehydrogenascs which catalyze direct proton transfer have sequential mechanisms (18) and many dis- play Ordered Bi-Bi kinetics (19, 20), it was concluded on the basis of the limited product inhibition studies and the calculated and experimentally determined K,, that glycerol dehydrogenase in either NH4+ or (C&,)4Nf catalyzes the reaction with an Ordered Bi-Bi mechanism.

The primary effect of monovalent cation activation is to de- crease the Michaelis constants for the substrates, particularly for glycerol and dihydroxyacetone (Table V and VI). The Michaelis constant for glycerol at pH 9.0 and pH 7.5 is 20 to 25 times larger when NH4f is replaced by (CH3CHz)4N+. For di- hydroxyacetone, the constant at pH 7.5 is 25 times larger but at 6.1 only 3 times larger when NH*+ is replaced by (CHBCH2)$J+. The constants for NAD or NADH are only 2-4 times larger at all pH values when NH4+ is deleted from the reaction. Glycerol increases the affinity for NH.,+ nearly 60-fold at pH 7.5. This nonsymmetric effect by monovalent cat,ions, that is, the effect on only one substrate of a two subst,rate reaction, has been observed in other systems. For formyltetrahydrofolate syn-

thetase, the K, for formate was decreased 10 to 12 times by NHb+, whereas the constant for ATP was not changed sig- nificantly (21). Similarly, for rat liver pyruvate carboxylase, the R, for HC03- was decreased lo-fold by K+ while the K, for ATP was decreased 2.2.fold (22).

An examination of the individual rate constants of Reaction 2 which may be calculated from the kinetic constants (15, 23) shows that the primary effect of the monovalent cation is on the rate constants, k3 and kg, for combination of glycerol or di- hydroxyacetone with their respective nucleotide binary complex to form the central complex. Since the incrcasc in these rate constants is much larger than the increase in V,,,,,, and since the affinity for NAD or NADII increases, which is usually reflected by a decrease in the off constants for the nucleotide, the increase in V,,, brought about by monovalent cation is probably due to an increased rate of interconversion of the central complex rather than release of nucleotidc.

The one question regarding monovalent cation activation which has evaded solution to date is whether or not monovalent cations activate enzymes by participating directly in catalysis

by promoting a change in the general over-all conformation or by both mechanisms. In the case of glycerol dehydrogenase, the first alternative, in which the monovalent cation is visualized to act as a bridge between glycerol and enzyme, is most con- sistent with the data. Such an interaction would be expected to decrease the K, for glycerol and dihydroxyacetone and have little or no effect on the K, or Ki, for NAD or NADH. The latter alternative, that is, a general conformational effect, would be expected to change the K, for all substrates.

Eisenman (24), after examining the sequence of affinities of five alkali cations, cesium, rubidium, potassium, sodium, and lithium, with a variety of biological and nonbiological materials noted that of the possible 120 permutations of this sequence, essentially only 11 are observed. He suggested that the key to the sequence specificity was the anionic field strength of the binding site. It is reasonable to expect that such anionic fields would be sensitive to pH; indeed, such an effect is observed.in ion exchange resins containing carboxyl groups, in which Naf is preferred over K+ at high pH but a lower pH caused reversal of the selectivity (25). Therefore, the observation that the sequence of affinities of monovalent cations with glycerol de- hydrogenase at pH 6.1, NHdf > Tl+ > K+ > Rb+, was dif- ferent from that at pH 9.0, NHd+ > Rb+ > Kf > Tlf, would be consistent with a change in the anionic binding site for the cation. The higher affinity for NH4+ would be consistent with a tetrahedral arrangement of the binding site.

Consideration of the general effects of monovalent cations on those enzyme systems which arc sensitive to them points to several observations (26-28). Monovalent cations (a) preferentially interact with one enzyme conformation, (6) often prevent protein subunit dissociation or protect against inactiva- tion, (c) generally reduce the K, for substrate, (d) often in- crease the maximum velocity, (e) vary in their abilities to acti- vate, with K+, Rb+, NHd+, and Tl+ usually being the most effective. The activation of glycerol dehydrogenase by mono- valent cations reflects these same points. First, NHd+, Rbf, K+, and Tl+ are the most effective activators; second, the activation follows Michaelis-Menten kinetics; third, a marked reduction in the K, for glycerol and dihydroxyacctone is ob- served; fourth, a 3- to 4-fold increase in maximum velocity is observed.

Acknowledgments--We thank Ms. Shirley Welch and Mr. Norman Young for assistance in computer programming and Professor E. C. C. Lin, Harvard University, for a gift of A. aerogenes strain 1033. We are deeply indebted to Dr. Joseph D. Shore for his valuable comments and criticisms.

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W. Glenn McGregor, James Phillips and Clarence H. SuelterAerobacter aerogenesGlycerol Dehydrogenase from

Purification and Kinetic Characterization of a Monovalent Cation-activated

1974, 249:3132-3139.J. Biol. Chem. 

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