ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 2, December,

AND BIOPHYSICS pp. 466-478, 1978

Mechanism of Action of a Wound-Induced w-Hydroxyfatty Acid:NADP Oxidoreductase Isolated from Potato Tubers (Solarium tuberosum L)’ VISHWANATH Department of Agricultural

P. AGRAWAL’

AND

P. E. KOLATTUKUDY3

Chemistky and Program in Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164 Received

June 26, 1978; revised

August

3, 1978

w-Hydroxyfatty acid:NADP oxidoreductase, an enzyme involved in suberin biosynthesis, is induced by wounding potato tubers. Initial velocity and product inhibition studies with the purified enzyme suggested an ordered sequential mechanism, where NADPH is added first, followed by 16-oxohexadecanoate, and NADP is released after 16-hydroxyhexadecanoate. Substrate inhibition by NADPH was observed at concentrations higher than 0.2 mre. The inhibitory NADPH molecule competes with 16-oxohexadecanoate, indicating that it forms a dead-end complex with the E-NADPH form of the enzyme. The kinetics for the NADPH inhibition suggested that n > 1 in the rate equation v = V[NADPH]/(K, + [NADPH] + [NADPH]n l ‘/ZQ; i.e., more than two NADPH molecules bind to enzyme. The K,,, for 16-oxohexadecanoate did not change from pH 7.5 to 9.0 but increased about lofold from pH 9.0 to 10.0, whereas the K, for NADPH and hexadecanal did not vary significantly in this pH range. Phenylglyoxal inactivated the enzyme; NADPH and AMP (which competes with NADPH; Ki = 1.1 mM) provided protection against such inactivation. Diethylpyrocarbonate also caused inactivation which was reversed by hydroxylamine; NADPH but not AMP protected the enzyme from this inhibition. Pyridoxal-5’-phosphate reversibly inactivated the enzyme and NaBH4 reduction of the pyridoxal phosphate-treated enzyme resulted in irreversible inhibition; a combination of NADPH and w-0x0 Cl6 acid provided protection against such inactivation. As the chain length of alkanals increased from Ca to CS, the K,,, for the substrate decreased drastically from 7000 to WpM and a further increase in chain length from Cs to Cm resulted in only a small decrease in K,,,. The K, and V for 8oxooctanoate and lo-oxodecanoate are compared with the values obtained for 16oxohexadecanoate. Based on these results, it is proposed that arginine acts as the binding site for NADPH, a hydrophobic crevice with lysine at the bottom forms the binding site for 16-oxohexadecanoate and histidine participates in the reaction as the proton donor.

Underground parts of plants are covered with suberin, which contains w-hydroxyfatty acid and dicarboxylic acid as the major classesof aliphatic monomers (1). Very little is known about suberin biosynthesis, except for the conversion of w-hydroxy acid to dicarboxylic acid, which is a two-step process involving w-oxoacid as an interme-

diate (2). The first step in this process is catalyzed by a wound-induced o-hydroxyfatty acid:NADP oxidoreductase which catalyzes the reversible oxidation of w-hydroxyfatty acid according to the following equation (3):

’ Scientific Paper 5128, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164. This work was supported in part by Grants PCM 74-09351 and PCM 77-00927 from the National Science Foundation. ’ Fulbright-Hays fellow from Tribhuvan University, Kathmandu, Nepal. 3 The author to whom all correspondence should be addressed.

The equilibrium of this reaction is in favor of the reduction of w-oxofatty acid. This dehydrogenase has been purified to near homogeneity from suberizing potato tuber disks and its preliminary characterization has been reported (3). The present paper describes initial velocity, and product and substrate inhibition

w-hydroxyfatty acid + NADP Z+ w-oxofatty acid + NADPH

466

0003-9861/78/1912-0466$02.00/O Copyright All rights

0 1978 by Academic F’reas, Inc. of reoroduction in anv form reserved.

+ H+

MECHANISM

OF

ACTION

OF

w-HYDROXY

studies, which suggest that this w-hydroxyfatty acid dehydrogenase prefers an ordered sequential mechanism. Based on the effect of pH and chain length of the substrates on K,,,, and chemical modification studies with phenylglyoxal, diethylpyrocarand pyridoxal-5’-phosphate, a bonate, model for the active site of w-hydroxyfatty acid dehydrogenase is proposed. MATERIALS

AND

METHODS

Chemicals. 16-Hydroxyhexadecanoic acid, phenylglyoxal, diethylpyrocarbonate, propanal, butanal, hexanal, o&anal, benzaldehyde, and borane-tetrahydrofuran complex were purchased from Aldrich Chemical Co. 16Cxohexadecanoic acid was synthesized as described before (3). Neral was isolated from citral (obtained from Aldrich Chemical Co.) by TLC on Silica Gel G with hexanediethyl ether (80:20, v/v) as the developing solvent. Pyridoxal-5’-phosphate, AMP, NADH, NADPH, and NADP were from Sigma Chemical Co. w-Hydroxyfatty acid:NADP oxidoreductase was purified from suberizing potato tuber disks as previously described (3). Clear dispersions of 16-hydroxyhexadecanoic acid and 16-oxohexadecanoic acid were prepared by sonication (3 x 5 s with the needle probe of Biosonik III at full power). Above 0.2 mM, turbidity was observed and therefore kinetic studies could not be performed with higher concentrations. Preparation of w-0x0 CS and GO acids. Monomethy1 ester of octane-1,8dioic acid and decane-l,lOdioic acid were prepared from the corresponding dicarboxylic acids (4), the half esters were reduced with BH3 (5) and the resulting w-hydroxy esters were saponified and oxidized with pyridinium chlorochromate (6). To 5 mmol of Cs half-ester dissolved in 10 ml freshly distilled tetrahydrofuran, 6 ml of BHa-THF (6 mmol) was added dropwise at 0°C with stirring. After an additional 10 min, excess BHs was decomposed with aqueous ethyl ether, the mixture was acidified with HCl, and extracted with ethyl ether. The ether layer was dried with sodium sulfate, evaporated to dryness, and the residual boric acid was removed by repeated volatilization with methanol. The residue was treated with 10 ml 1 M KOH in 99% methanol for 16 h at room temperature. After addition of water to dissolve the precipitate, the solution was extracted with ethyl ether, acidified with HCl, and the product was recovered by extraction with ethyl ether. 8-Hydroxyoctanoic acid was purified by TLC on Silica Gel G with diethyl ether:hexane:formic acid (30~20~1, v/v) as the developing solvent and identified by combined gas-liquid chromatography-mass spectrometry. The diagnostic fragments of the trimethylsilyl derivative were: M’-CR, m/e 289, 93% of the base peak; M+CH&H,, m/e 273, 31%; and M’-CHZ-(CH&SiOH, m/e 199, 69%. Metastables corresponding to M-CHZ + M+-CH3-CH4 and M’-CH3 - M+-CH&CH&SiOH

ACID

DEHYDROGENASE

467

were also observed. 8-Oxooctanoic acid was prepared from 8-hydroxyoctanoic acid by oxidation with pyridinium chlorochromate as described before (3). The product was purified by TLC with the above solvent system and identified by combined gas-liquid chromatography-mass spectrometry. The diagnostic fragments of the trimethylsilyl ester derivative were M’CH3, m/e 215, 20% of the base peak; M’-CHVHZO, m/e 197, 39%; M+-CHs-CO, m/e 187, 42%; M+-CHZCH20, m/e 185,28%; and M+-CHs-CHCHO, m/e 171, 19%. The metastables corresponding to transitions MCH3 --) M+-CHJ-H*O and M+-CH3 + M+-CH,-CO were also observed. The identification of the oxoacid was verified by NaBHa reduction followed by gasliquid chromatography-mass spectrometry of the resulting 8-hydroxyoctanoic acid. lo-Oxodecanoic acid was prepared from the corresponding half-ester and identified as described above for the 8-oxooctanoic acid. Aqueous dispersions of Ca and Cl0 w-oxoacids were prepared by sonication as indicated above. Preparation of C,OG Aldehydes. CIO-G4 aldehydes were prepared by stirring the corresponding alcohols in CH&h with pyridinium chlorochromate for 1 h at room temperature and the product was purified by TLC on Silica Gel G with hexane-diethyl ether (80:20, v/v) as the developing solvent. Aqueous dispersion of the aldehydes were prepared by sonication as described above. Initial velocity measurements. The reaction was followed by measuring the appearance or disappearance of NADPH as indicated by absorbance at 340 nm measured with a Beckman model 25 K spectrophotometer. The cell compartment was thermostatically controlled at 30°C by electrical heating. The cuvette, ffied with appropriate reaction components, was brought to 30°C and the reaction was initiated by addition of the enzyme. The amounts of enzyme used were selected to give linear reaction rates for 3 to 5 min and only the initial velocities were used in all experiments. For all kinetic measurements, unless otherwise indicated, the incubation mixture contained 0.4 M glycine-NaOH buffer, pH 8.5 in a total volume of 0.25 ml. Treatment of kinetic data. The reciprocals of velocities were plotted against the reciprocals of the substrate concentrations; the least square method for straight line was used to fit the data. Double reciprocal plots of initial velocity and product inhibition data were examined to determine the pattern (i.e., intersecting, competitive inhibition, etc.) according to Cleland (7) and the slopes (k/v) and intercepts (l/v) were plotted against either the reciprocal of the concentration of the nonvaried substrate (for initial velocity experiments) or the inhibitor concentration (for product inhibition experiments) to determine the linearity of these replots. Kinetic constants and product inhibition constants were obtained from the secondary plots (7). For all calculations, except for substrate inhibition, the following general rate equation for a two substrate-two product reaction was used (7):

466

AGRAWAL VAB

’ = Ki.Kb + K.B + KbA + AB

AND (1)

In this equation, v is the observed velocity, A and B are substrate concentrations, V is the maximum velocity, K. and Kb are the Michaelis constants, and K, is the dissociation constant of the enzyme-A complex in a sequential mechanism. Treatment of the enzyme with phenylglyoxal. To determine concentration dependence, 1 pg enzyme (10 al) was incubated with 10 ~1 of phenylglyoxal solution in 0.2 M phosphate, pH 8.3, for 10 min at 30°C and 5 ~1 of this mixture was assayed for enzyme activity. For determination of time-course of inactivation, 3 ag enzyme (30 d) was similarly incubated with 30 al of 10 mru phenylglyoxal solution and 5-1.11 aliquots were assayed for enzyme activity at desired intervals. The enzyme assay mixtures (250 4) contained phosphate buffer instead of glycine-NaOH buffer. Treatment with diethylpyrocarbonate. For timecourse experiments, 20 d of 0.1 or 0.01 M diethylpyrocarbonate solution in ethanol was added to 5 ag enzyme in 180 4 of 0.14 M phosphate buffer, pH 6.5, and the mixture was incubated at 30°C; lo-al aliquots were assayed for enzyme activity at desired intervals. Treatment withpyridoxaZZ’-phosphate. For determination of concentration dependence, 1 ag enzyme (10 ~1) was incubated with 10 4 of pyridoxal-5’-phosphate solution in 0.2 M phosphate buffer, pH 8.0, at 30°C for 30 min and 5 al of this mixture was assayed for enzyme activity. For the determination of timecourse of inactivation, 3 pg enzyme (30 al) was similarly incubated with 30 al of 20 mM pyridoxal-5’-phosphate solution and 5-d aliquots were assayed for enzyme activity at desired intervals. Enzyme assays. In the case of chemical modification studies with phenylglyoxal, diethylpyrocsrbonate, and pyridoxsl phosphate, small (5-10 4) aliquots of the reaction mixtures were assayed for dehydrogenase activity with 40 pM 16-oxohexadecanoate and 80 pM NADPH in a total volume of 0.25 ml of 0.4 M glycineNaOH buffer, pH 8.5, unless otherwise specified. The extent of protection by substrates and analogues was determined by adding the test compound prior to the addition of the modifying agent and protection is expressed as the enzyme activity retained due to the presence of the protector/activity lost in the absence of the protector. In substrate specificity studies with the different 0x0 compounds, NADPH oxidation was followed spectrophotometrically as above, and K, and V were determined from direct linear plots (81. Chromatography. Thin-layer chromatography was performed on l-mm Silica Gel G layers (20 x 20 cm) which were activated overnight at 110°C. Compounds were visualized under UV light after spraying the plates with 0.1% ethanolic solution of 2,7-dichlorofluorescein. Combined gas-liquid chromatography-mass spectrometry was performed with a column temperature of 160°C as described earlier (3).

KOLATTUKUDY RESULTS

Initial

AND

DISCUSSION

Velocity Studies

To determine the order of binding of the substrates to the enzyme initial velocities in both the forward and the reverse directions were measured with varying concentrations of one substrate at fixed concentrations of the other. Double reciprocal plots with 16-oxohexadecanoate as the variable substrate at fixed concentrations of NADPH revealed an intersecting pattern (Fig. 1A). With NADPH as the variable substrate at fixed concentrations of 16oxohexadecanoate an intersecting pattern (Fig. 1B) was again observed. In the forward direction also, double reciprocal plots with 16-hydroxyhexadecanoate or NADP as the variable substrate showed intersecting patterns (Fig. 1, C and D). The kinetic constants calculated from the replots of slopes and intercepts (Fig. 1, insets) are listed in Table I. The intersecting nature of the double reciprocal plots obtained from initial velocity studies for both the forward and the reverse reactions strongly suggests a sequential mechanism for the reaction (2), by ruling out a ping-pong mechanism which would have given parallel patterns in the double reciprocal plots. The Km for NADPH was 40% of that for NADP, suggesting that NADPH binds to the enzyme more tightly than does NADP. Several other dehydrogenases also show higher affinity for the reduced pyridine nucleotide than for the oxidized form (9).

Product

Inhibition

Studies

To determine the order of addition of substrates and release of products, product inhibition studies were undertaken. Since the rate in the reverse direction was comparatively fast, and hence easily measurable, these studies were conducted on the reverse reaction. Initial velocities were measured with varying concentrations of one substrate at fixed concentrations of a product, keeping the concentration of the other substrate fixed. With NADP as the inhibitor and 16-oxohexadecanoate as the variable substrate, a linear, noncompetitive inhibition pattern was obtained (Fig. 2A). On the other hand, with NADP as the inhibitor and NADPH as the variable substrate, a linear competitive inhibition pat-

MECHANISM

OF

ACTION

OF

w-HYDROXY

V[I~-OXOHEXADECANOATE](I/C~M)

r

FIG, 1. Double reciprocal plots for the forward and acid:NADP oxidoreductase. Concentrations (PM) of canoate (B), NADP (C), and 16-hydroxyhexadecanoate Amounts of enzyme used were 0.962 cog for A and B, rate was measured at pH 9.5 and reverse reaction intercept.

A. Kinetic

PARAMETERS constants

OF GJ-HYDXOXYFATTY

obtained Forward

from

initial

reaction

(PM)

reverse reactions catalyzed by w-hydroxyfatty fmed substrates, NADPH (A), l&oxohexade(D) were as indicated in the figure. and 0.62 pg for C and D. The forward reaction at pH 8.5. Insets show replots of slope and

constants

obtained

LI K,i and ’ Equations K,i and

from

FROM

product

Variable

SUBEXIZING

POTATO

exneriments”



.

inhibition

reaction

(PM)

KNADPH Ku-cm cl6 acad K 1NADPH

90.2 + 2 27.2 f 0.2 56.8 f 0.1

Inhibitor NADP NADP w-Hydroxy acid w-Hydroxy acid

OXIDOXEDUCTASE

Backward

KNADP Ktio-hysaoxy c,~ acid K I NADP B. Inhibition

I

ACID:NADP DISKS

velocitv

469

DEHYDROGENASE

I/[NADPH](I/pM)

TABLE KINETIC

ACID

5.9 + 0.2 8.3 f 0.4 3.5 + 0.1

experiments”

substrate

Pattern

Constant

(PM)

KLi

KLS

Cl6

NADPH w-0x0 Cl6 acid NADPH

Competitive Noncompetitive Noncompetitive

200 274b

14.4 120 9006

C,6

w-0x0

Noncompetitive

300*

260b

K, are inhibition [interceptlr

K,, respectively,

constants = [interceptlo

where

subscripts

Cl6 acid calculated

from

intercept

and slope replots,

respectively. were

used to calculate

470

AGRAWAL

AND

tern was obtained (Fig. 2B). The product inhibition caused by 16-hydroxyhexadecanoate at lower than 0.1 mu were too low to measure accurately and concentrations higher than 0.1 nuu could not be reached because of poor solubility. Therefore, experiments with 16-hydroxyhexadecanoate were performed only at 0.1 mM. Double reciprocal plots with 16-hydroxyhexadecanoate as the inhibitor and 16-oxohexadecanoate or NADPH as the variable substrate showed noncompetitive patterns (Fig. 2, C and D). The results obtained from product inhibition studies are summarized in Table I. Since NADP competes with NADPH, both NADPH and NADP probably bind to the same form of the enzyme. Noncompetitive inhibition by the product NADP

I/116-OXO~XPSE~NE](l/~M]

8 6 4 2 :”

1416-0X0

0

I/[NADPH]

WpM]

C,sACID](l/fi)

0.1

I/INADf+il

KOLATTUKUDY

against 16-oxohexadecanoate suggests that NADP and 16-oxohexadecanoate bind to different forms of the enzyme in equilibrium with each other. These results suggest that w-hydroxyfatty acid dehydrogenase, under the present experimental conditions, prefers an ordered sequential mechanism where NADPH binds to the enzyme first followed by w-oxofatty acid and NADP is released after w-hydroxyfatty acid (Fig. 3). This conclusion is supported by the observed noncompetitive inhibition by 16-hydroxyhexadecanoate against NADPH. The noncompetitive inhibition by 16-hydroxyhexadecanoate with 16-oxohexadecanoate as the variable substrate strongly suggests the presence of one or more central complexes and rules out a mechanism of Theorell-Chance type according to which 16-hydroxyhexadecanoate would be a competitive inhibitor when 16-oxohexadecanoate is varied (7). A random mechanism is also inconsistent with the present results as such a mechanism would have given at least two competitive patterns for product inhibition (7). Even though the present results, in favor of the sequential ordered mechanism, were obtained only for the reverse reaction, it is likely that they apply for the forward reaction as well. The ordered sequential mechanism proposed for the present enzyme is similar to those proposed for several other dehydrogenases such as lactate dehydrogenase (lo), alcohol dehydrogenase (ll), malic enzyme (12), malate dehydrogenase (13), ribitol dehydrogenase

D 0.2

0.3

E-(NADPWn,,

W,.M)

FIG. 2. Product inhibition (of reverse reaction calta lyzed by w-hydroxyfatty acid:NADP oxidoreductase. Inhibition by NADP with 16-oxohexadecanoate (A) and NADPH (B) as the variable substrates and by 16hydroxyhexadecanoic acid with 16-oxohexadecanoic acid (C) and NADPH (D) as the variable substrates are shown. Concentrations of fixed substrates, NADPH (A and C) and 16-oxohexadecanoate (B and D), were 40 and 32 PM, respectively. NADP concentrations (PM) were as given in the figure. The concentration of w-hydroxyacid in C and D was 106 PM. Amount of enzyme used was 0.125 ).q. In C and D: A, with ohydroxyhexadecanoate; 0, without the w-hydroxy acid.

FIG. 3. The order of addition of substrates and release of products in the reaction catalyzed by whydroxyfatty acid:NADP oxidoreductase from suberixing potato tuber disks.

MECHANISM

OF

ACTION

(14), and glyceraldehyde-3-phosphate hydrogenase ( 15).

Inhibition

OF

w-HYDROXY

de-

by AMP

AMP is known to compete with the pyridine nucleotide binding site of several dehydrogenases (9). To determine if AMP is a coenzyme analogue for the present whydroxyfatty acid dehydrogenase, reaction rates with NADPH as the variable substrate, at different concentrations of AMP, were measured. Double reciprocal plots yielded a linear competitive inhibition pattern (data not .shown) indicating that AMP might be useful in exploring the coenzyme binding site as it competes with NADPH for the same site (Ki = 1.1 mM).

Substrate

Inhibition

by NADPH

ACID

DEHYDROGENASE

471

hibition by higher NADPH concentrations is graphically illustrated by a double reciprocal plot which shows a linear relationship up to 0.1 lll~ NADPH, and above 0.2 mM, a sharp upward curve is observed (Fig. 4). To determine whether one or more than one molecule of NADPH binds to the ENADPH form of the enzyme, v was plotted against -log[NADPH] (Fig. 4). If n in the above equation were equal to one, a symmetrical bell-shaped curve would have resulted from such a plot (6). However, in the present case, an unsymmetrical plot was obtained with v = 0 at a value of 3 for -log[NADPH] (Fig. 4). The steeper fall in velocity in the higher NADPH concentration range probably results from the binding of more than one NADPH molecule to the E-NADPH complex, i.e., the value of n > 1 in the above rate equation (16). The binding of the inhibitory NADPH molecule(s) to the enzyme (E-NADPH or some other form) might induce a conformational change leading to a new form of the enzyme which has either a high Km for the w-oxofatty acid or a less efficient catalytic site or both. Substrate inhibition by noncoenzymic substrate is common among dehydrogenases (9) but inhibition by coenzyme is less common. One example, where a dehydrogenase is inhibited by reduced coenzyme (as the substrate), is the case of glutamate dehydrogenase which is inhibited by NADH. In this case, a symmetr-

During the initial velocity studies, it was noticed that the reaction was strongly inhibited by NADPH at concentrations greater than 0.2 mM. To determine the mechanism of NADPH inhibition, initial velocities were measured at varying concentrations of 16-oxohexadecanoate with inhibitory concentrations of NADPH. Double reciprocal plots (Fig. 4) gave a linear competitive pattern, suggesting that both w-oxofatty acid and the inhibitory molecule of NADPH bind to the same form of the enzyme. Thus, it appears that NADPH forms a dead-end complex by binding to the E-NADPH form of the enzvme. However, the possibility that NADPH also binds to other forms of the enzyme cannot be ruled out. To determine the rate equation explaining the kinetics of the NADPH inhibition, this substrate inhibition may be represented by E + NADPH + E-NADPH; ENADPH + n NADPH = E-(NADPH), +1 (n 1 1). From these equations, it follows that v/V = l/El + K,/[NADPH] + [NADPH]“/Ki]; when n = 1, one inhibitory molecule of NADPH binds to E-NADPH giving E-(NADPHh complex which is not involved in the catalytic sequence (deadend complex). To study the kinetics of F1c.4.Substra ’ . ‘. ’ . ot^ the. reaction catalyzed te ~hlbltlon NADPH inhibition, initial velocities were by w-hyhOxyfat ,ty acid:NADP oxidoreductase by measured with varying concentrations of NADPH. cancer Itration of 16-oxohexadecanoate was NADPH (from 1 pM to 1 m&4) at one fixed kept fixed at 32 pM (A and B) and 0.125 pg enzyme concentration of 16-oxohexadecanoate. Inwas- used.

472

AGRAWAL

AND

ical curve for v us -log NADH plot was observed and it was postulated that with excess NADH, two molecules of NADH bind to the NADH binding site (17). With this enzyme, substrate inhibition was caused specifically by NADH but not by NADPH, whereas with the present w-hydroxy acid, dehydrogenase both NADH and NADPH exhibited substrate inhibition. Effect of pH on K,,, To obtain information about the nature of the amino acids involved in substrate binding, the effect of pH on the K,,, of 16oxohexadecanoate and NADPH was studied (Fig. 5). The K, for 16-oxohexadecanoate (at 80 PM NADPH), which did not change in the pH range of 7.5 to 9, increased drastically as the pH changed from 9 to 10. On the other hand, K, for NADPH (at 40 j.bM 18oxohexadecanoate) did not change in this entire pH range (7.5 to 10). These results strongly suggest that a protonated positively charged group with a pk, in the range of 9 to 10 may be involved in binding l&oxohexadecanoate to the enzyme-coenzyme complex. If this group binds the w0x0 Cl6 acid through an ionic interaction with the carboxyl group of the substrate, substrate analogues without the carboxyl group should bind to the enzyme less tightly (high K,) and the K, of such analogues should not increase as the pH increases Ia-

I w-OXOCle

5 a :

ACID v’

80. HEXADECANAL 40.

O

:

I

7

1

7.5

I 0.0

I 8.5

I 9.0

I 9.5

I I IO

PH 5. Effect of pH on the Zf,,, of w-hydroxyfatty acid:NADP oxidoreductase for NADPH, 16-oxohexadecanoate, and hexadecanal. FIG.

KOLATTUKUDY

from 9 to 10. In fact, hexadecanal, which is structurally identical to the 16-oxoacid, except for the absence of the carboxyl group, showed a nearly pH-independent K,,, which was higher than that of the o-0x0 Cl6 acid (Fig. 5). These results suggest that the Eammonium group of lysine might be involved in w-0x0 Cl6 acid binding through an ionic interaction with the carboxyl group. The pH independence of K,,, for NADPH from 7.5 to 10 suggests that the pK, of the ionizable group, if any, involved in the coenzyme binding lies outside this pH range. The K, for lo-oxodecanoic acid (55 PM), which was substantially higher than that of the Cls w-0x0 acid (12 PM), did not increase as the pH was changed from 8.5 to 10, indicating that the e-ammonium group of the lysine residue does not interact with the carboxyl group of the shorter substrate. Chemical Modification For obtaining information about the catalytic mechanism of w-hydroxyfatty acid:NADP oxidoreductase, amino acid modification studies were undertaken to explore the active site of this enzyme. Arginine modification with phenylglyoxal. Many enzymes with negatively charged substrates, including dehydrogenases, are known to be inhibited as a result of modification of arginine residues (18-20). In the dehydrogenases the guanidino group of arginine apparently interacts with the pyrophosphoryl group of the coenzyme. To determine if such an interaction is involved in w-hydroxyfatty acid dehydrogenase, phenylglyoxal, which is known to specifically react with the guanidino group of arginine under mild conditions (21), was used for arginine modification. Reduction of 16oxohexadecanoic acid catalyzed by the enzyme was inhibited by phenylglyoxal in a concentration-dependent manner; at 5 mu phenylglyoxal, 60% inhibition was observed in 10 min (Fig. 6A). Furthermore, 5 mM phenylglyoxal inactivated the enzyme in a time-dependent fashion, suggesting that arginine residue(s) is essential for the enzyme activity. If this inhibition is caused by modification of arginine(s) present in the active site, substrates or analogues might protect the enzyme from inactivation. The presence of NADPH (5 mu) or NADP (5 mu) af-

MECHANISM

OF

ACTION

OF

w-HYDROXY

ACID

473

DEHYDROGENASE

about 43% protection. On the other hand, the presence of o-oxohexadecanoate, (0.5 mu) in the reaction mixture did not affect the extent of inactivation of the enzyme. These results strongly suggest that arginine(s) is present in the active site and serves as the binding site for NADPH. This

forded about 60% protection against inactivation by phenylglyoxal (Table II). If the protection by the pyridine nucleotides is due to the binding of the phosphate to the guanidino group, AMP, which competes with NADPH, might be expected to protect the enzyme; in fact, 5 mu AMP provided

10 20 0 TIME (MINI

PLP (mM1

20 40 TIME (MINI

FIG. 6. Inactivation of w-hydroxyfatty acid:NADP oxidoreductase activity by phenylglyoxal (A) by diethylpyrocarbonate (B) and by pyridoxal phosphate (C). Experimental details are described in the text. DEPC, diethylpyrocarbonate and PLP, pyridoxal phosphate. In B, the recovery of the enzymic activity 5 min after the addition of 0.1 M hydroxylamine is indicated by the dashed line. TABLE PROTECTION

OF W-HYDROXYFATTY

ACID:NADP MODIFYING

II

OXIDOREDUCTASE

Treatment 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

FROM

INACTIVATON

BY CHEMICAL

AGENT.!?

Enzyme aIone + 5 mM phenylglyoxal + 5 mM phenylglyoxal + 5 mM NADPH + 0.5 mM 16-oxohexadecanoate + 5 mM phenylglyoxal + 5 mM AMP + 5 mu phenylglyoxal + 5 mru NADP + 5 mM phenylglyoxal + 10 mM diethylpyrocarbonate (DEPC) + 1 mM NADPH + 10 mu (DEPC) +5mbfAMP+lOmMDEPC + 0.5 mM 16-oxohexadecanoate + 10 mM DEPC + 0.5 mM 16-oxohexadecanoate + 1 mM NADPH + 10 m&f DEPC + 10 mM DEPC + 0.1 M hydroxylamine, pH 7 + 10 mM pyridoxaW-phosphate (PLP) + 10 mu PLP (dialysed) + 10 mM PLP + NaBH, (dialysed) + 5 mM AMP + 10 mM PLP + 0.5 mM 16-oxohexadecanoate + 10 mM PLP + 5 mu NADPH + 10 mM PLP + 5 mM NADPH + 0.5 mu 16-oxohexadecanoate + 10 mM PLP + 0.1 mM p-hydroxymercuribenzoate + 0.1 mM p-hydroxymercuribenzoate + 5 mru DTE + 5 mu NADPH + 0.1 mu p-hydroxymercuribenzoate

Relative rate of control)

(%

loo 9 61 10 48 66 4 78 25 10 85 80 31 90 10 25 32 60 76 6 9 7

a Incubation involving phenylglyoxal, diethylpyrocarbonate, pyridoxab5’-phosphate, and p-hydroxymercuribenzoate were done at pH 8.3, 6.5, 8.0, and 8.5, respectively for 30, 5, 30, and 10 min, respectively. Enzyme assays were done as described in the text. All values are based on appropriate controls. Experimental details are described in Materials and Methods.

474

AGRAWAL

AND KOLATTUKUDY

conclusion is supported by the observation that K,,, for NADPH stays virtually constant in the pH range of 7.5 to 10. This result also rules out the involvement of the e-ammonium group of lysine in the binding of NADPH because, if it were involved, the Km for NADPH would have significantly changed as the pH was changed from 9 to 10. This conclusion is particularly relevant in view of the possibility that phenylglyoxal can react with e-amino group of lysine if treated with this reagent for a long period (21). The possibility that arginine is involved in w-oxoacid binding appears to be unlikely as the K,,, for ~0x0 C16 acid increased sharply when the pH changed from 9 to 10. Histidine Modification Diethylpyrocarbonate

with

Several enzymes including dehydrogenasesare inhibited by diethylpyrocarbonate, which specifically carbethoxylate histidines at pH 6.0 (22). The precise role of histidine(s) in dehydrogenases is not well understood except in lactate dehydrogenase where it serves as an acid-base catalyst (23). In horse liver alcohol dehydrogenase, histidine residues do not appear to be involved in catalysis but are reported to be important for maintaining the tertiary structure of the enzyme (24). To determine the role of histidine, if any, in w-hydroxy acid dehydrogenase, the effect of diethylpyrocarbonate on enzymic activity was studied. Since the w-hydroxy acid dehydrogenase activity was very unstable at pH 6 or below, modification studies with this reagent were carried out at pH 6.5. The time course of inactivation of w-hydroxy acid dehydrogenase at 1 and 10 mru diethylpyrocarbonate showed that at both concentrations, there was a rapid decrease in activity followed by a slower rate of inactivation (Fig. 6B); in 5 min, there was virtually complete loss of enzymic activity at 10 loll diethylpyrocarbonate. Treatment of the inactivated enzyme with hydroxylamine, which is known to regenerate histidine from its carbethoxy derivative (22), resulted in about 72 to 88% recovery of the enzymic activity within 5 min (Fig. 6B). These results, along with the known specificity of diethylpyrocarbonate for histidine in a variety of proteins,

strongly suggest that diethylpyrocarbonate probably modified histidine(s) necessary for the activity of w-hydroxy acid dehydrogenase. The enzyme was protected (nearly 80%) by 1 II~M NADPH from inactivation by diethylpyrocarbonate but w-oxohexadecanoate and AMP afforded very little protection (Table II). The observation that AMP, which competes with NADPH, provided very little protection from inactivation by diethylpyrocarbonate suggests that histidine is most probably not involved in binding of the phosphate moiety of NADPH. The protection by NADPH (but not by AMP) suggests that histidine, in the active site, is probably in close proximity to the nicotinamide ring of NADPH and this structural feature is involved in the protection of histidine by NADPH from diethylpyrocarbonate modification. This close proximity would be required for histidine to function as a proton donor and the observed complete inactivation by diethylpyrocarbonate is consistent with such a functional role for histidine in catalysis. The possibility of modification of an essential lysine residue located in a special pK,-lowering environment, such as that postulated in the case of glutamate dehydrogenase, cannot be ruled out in the present case (25). Lysine Modification phosphate

with PyridoxaL-B-

PyridoxsW-phosphate has been used to specifically modify lysine through Schiffs base formation in a reversible manner in a variety of enzymes (26). In view of the conclusion reached from the pH dependence of Km that the e-ammonium group of a lysine residue might be involved in w-0x0 Cl6 acid binding, the effect of pyridoxal phosphate on the enzyme activity was examined. Pyridoxal phosphate inhibited the dehydrogenase in a time and concentration dependent fashion (Fig. 6C); in 30 min, 10 mM pyridoxal phosphate inactivated the enzyme by 70%. If pyridoxal phosphate inactivated the enzyme by forming Schiffs base with lysine, the inactivation might be reversed by dialysis, whereas sodium borohydride would irreversibly inactivate the pyridoxal phosphate-treated enzyme by reducing the Schiff’s base. In fact, dialysis of

MECHANISM

OF

ACTION

OF

w-HYDROXY

the pyridoxal phosphate-treated enzyme (70% inhibited) restored the bulk (86%) of the enzyme activity, whereas NaBH4 treatment of the pyridoxal phosphate-treated enzyme prior to dialysis resulted in nearly complete loss of enzymic activity (Table II). To determine the role of lysine in this enzyme, protection experiments were carried out. The presence of 5 m NADPH in the incubation mixture afforded over 40% protection against inactivation by pyridoxal phosphate, whereas AMP or 16-oxohexadecanoate alone did not affect the extent of inactivation but the oxoacid in combination with NADPH provided 65% protection. The e-ammonium group of lysine could serve as the positively charged group, which on the basis of the pH effect on K,, was postulated to be involved in the ~0x0 Cl6 acid binding. The failure of the w-0x0 Cl6 acid residue (alone) to protect the enzyme and the ability of this substrate to significantly enhance the protection afforded by NADPH alone are consistent with the ordered squential mechanism postulated on the basis of kinetic studies and with the hypothesis that the distal carboxyl group of the ~0x0 Cl6 acid interacts with the e-ammonium group of lysine. Protection by NADPH alone against pyridoxal phosphate might be because it binds the arginine residue and thus blocks the access of pyridoxal phosphate to the lysine residue, which is postulated to be present in a crevice at the w-oxoacid binding site. If lysine were involved in NADPH binding, AMP should have protected the enzyme from inactivation by pyridoxal phosphate and the K,,, for NADPH should have been sensitive to change in pH in the range of 9 to 10, contrary to the results presented in a previous section. The failure of increasing concentrations of pyridoxal phosphate to lead to complete inactivation might be caused by its binding to a second site, such as the arginine residue, located near the opening to the crevice thus decreasing the access of pyridoxal phosphate to the lysine located at the bottom of the crevice.

Effect of other inhibitors Incubation of the enzyme with 0.1 mM phydroxymercuribenzoate for 10 min almost

ACID

475

DEHYDROGENASE

completely (95%) inactivated the enzyme (Table II). Treatment of inactivated enzyme with dithioerythritol did not restore the activity. Lack of protection by NADPH against this irreversible inactivation suggested that the amino acid residue(s) modified by p-hydroxymercuribenzoate lie outside the active site and somehow its modification irreversibly disrupts the conformation needed for the activity. Metal chelating agents such as l,lO-phenanthroline, bipyridyl, and EDTA had no effect on enzyme activity, suggesting that this dehydrogenaseprobably does not require metal ions for activity.

Evidence for a Hydrophobic Region the Substrate Binding Site

in

The observation that hexadecanal was a good substrate for the w-hydroxyfatty acid dehydrogenase indicated the presence of a hydrophobic region in the substrate binding site. If this is true, aliphatic aldehydes with very short carbon chains would be expected to have low affinity for the enzyme. In fact, acetaldehyde showed no measurable activity and propanal showed very little activity with an extremely high &,. As the chain length increased from Cs to CS, K,,, decreased drastically (7000 to 90 pM) and further increase in chain length from CSto Go resulted in only a small decrease in Km (Fig. 7). The observation that the smallest substrate which has a reasonably high affinity for the enzyme (Km < 100 PM) is octanal, suggests the involvement of hydrophobic interaction in substrate binding. In view of the observation that octanal showed a nearly maximal affinity for the enzyme, we used 8-oxooctanoic and lo-oxodecanoic acids as substrates to determine the approximate number of methylene groups required for optimal binding of w-oxoacids. The K,,, for lo-oxodecanoic acid (55 PM) was nearly the same as those obtained for Cs and longer aldehydes, whereas Km for 8oxooctanoic acid was substantially higher (Km, 310 ,uM; V, 928 nmol/min) and close to that of hexanal. These results indicate that the minimum number of methylene groups required for optimal binding of w-oxoacids is eight. The decrease in the hydrophobicity caused by the replacement of the methyl group of octanal by a carboxyl group in-

476

AGRAWAL

f Km

AND

-I

ALDEHYDE CHAIN LENGTH FIG. 7. Effect of the chain length of alkanals on their K,,, and V for w-hydroxyfatty acid:NADP oxidoreductase. K,,, and V were determined at 80 PM NADPH. Relative V is expressed as a percentage the V (2.21 pmol/min/mg) for 16-oxohexadecanoate.

of

creased the K,,, 3- to 4-fold, whereas the introduction of a carboxyl group with two additional methylene groups in the aliphatic chain (lo-oxodecanoic acid) resulted in optimal binding. The K,,, for lo-oxodecanoic acid was independent of pH in the range of 8-10 (data not shown), suggesting that the e-ammonium group of lysine, postulated to be involved in the binding of 16oxohexadecanoate, does not participate in the binding of the shorter w-oxoacid. Most probably lo-oxodecanoic acid is bound to the hydrophobic region close to the active site but it is too short to interact with the lysine residue. The binding of 18oxohexadecanoate to the enzyme most probably involves both the hydrophobic interaction, which bring the K, to the range of 40 to 100 PM, and the ionic interaction, which probably further lowers the K, to about 10 PM. With aliphatic aldehydes as substrates, the decrease in K, observed as the chain length increased from CS to Cg was accompanied by a drastic increase in V, but further increase in chain length resulted in a sharp decrease in V up to C12. Further increase in chain length up to Cl8 showed little effect on V, whereas Czogave less than one-third of this value and CL2and higher

KOLATTUKUDY

aldehydes did not even show detectable rates. The reasons for these changes in V are not understood. However, it appears possible that the rate-limiting step is the addition of the substrate to the E-NADPH form of the enzyme for C&S aldehydes, whereas for longer substrates, due to their increased hydrophobic interaction with the enzyme, release of the product from the ternary complex becomes rate limiting. This interpretation is also supported by the observation that lo-oxodecanoic acid showed a substantially (3.8 times) higher V than that obtained with 16-oxohexadecanoic acid. The drastic drop in V for aldehydes longer than Cl8 is most probably because the hydrophobic crevice is not large enough to accommodate alkyl chains much larger than Gs. The observation that very long carbon chains (>CB) are found in the o-hydroxy acid fraction but not in the dicarboxylic acid fraction of suberin from potatoes and other plants was previously postulated to be due to the inability of whydroxy acid dehydrogenase to oxidize such very long substrates (27, 28). The present results with the isolated enzyme provide direct evidence in support of the above postulate and such a substrate specificity appears to be conferred on the enzyme by a limitation in the depth of the hydrophobic crevice. In view of previous reports that extracts of potato tubers contain separate dehydrogenasesspecific for aliphatic, aromatic, and terpene alcohols (29, 30) we examined whether the present wound-induced dehydrogenase would fit into one of these categories. The present purified enzyme catalyzed reduction of both benzaldehyde (Km, 400 PM; V, 1.66 ~mol/min/mg protein) and neral (K,,,, 70 PM; V, 354 nmol/min/mg protein). It appears that any alcohol with enough hydrophobicity to interact with the hydrophobic crevice of the active site can be a substrate for this enzyme and no clear preference for aliphatic, aromatic, and terpene alcohols is obvious. In any case, the properties of the present wound-induced enzyme do not appear to be similar to any of the previously examined dehydrogenases from potato. Comparison of V/K,,, values. Compari-

MECHANISM

OF

ACTION

OF

w-HYDROXY

son of V/K, values for alkanals of varying chain length suggested a high specificity for Cs to Cl8 substrates (Table III). Even though these values were obtained from measurement on the reaction in the reverse direction, V/K,,, for substrates for the forward reaction should be proportional to the V/K, for reverse reaction provided Kes and K rNA~~/Kt~A~~~ do not alter substantially with change in chain length of the substrate. This reasoning is based on the Haldane equation:

For the redox pair alkanal/alcohol, Keg is likely to be virtually independent of the chain length. K~NADP/K~NADPH &O should not change with chain length of the aliphatic substrate as the kinetic studies indicated that the coenzyme is the first substrate to bind (31). Based on the results obtained from chemical modification of w-hydroxyfatty acid dehydrogenase, the model shown in Fig. 8 is proposed for the active site of the enzyme. According to this model, an arginine residue K,

AND V OF U-HYDROXYFATTY

Substrate

A. Alkanals CO CB Cd C6 CS GO Cl2 CM Cl.5 C18 CXl C22 C24 B. w-0x0 acid 8-0~0 10-0x0 16-0x0

TABLE AcID:NADP Km (PM)

ACID

in the active site binds to NADPH through interaction of its guanidino group with the pyrophosphate moiety of NADPH. The binding site for 16-oxohexadecanoate is

ADENOSINE-O-p-O-P-O-RIB

FIG. 8. A proposed model for the active site of whydroxyfatty acid:NADP oxidoreductase from woundheahng potato tuber disks. III OXIDOREDUCTASE

FOR 0x0

V (nmol/min/mg)

SUBSTRATES VI&

-*

-b

Cs acid GO acid Cl6 acid

477

DEHYDROGENASE

7ooo 1800 292 88 65 45 38 50 62 71 -b -*

287 906 1724 5525 3536 1879 1724 1768 1503 641 -* -*

310 55 12

8376 2210

460 70

1658 354

928

0.04 0.50 5.9 63.14 54.40 41.64 45.37 35.36 24.24 9.03

2.79 152.3 184.2

C. Other substrates BenxaIdehyde Neral 0 Experimental details are described * K, and V could not be determined

in Materials due to very

and Methods. low rates with

these substrates.

4.14 5.06

478

AGRAWAL

AND

composed of a hydrophobic crevice which interacts with the aliphatic chain and a lysine residue (located at the bottom of the crevice) which interacts with the carboxvl group of the ~0x0 Cl6 acid. Histidine p& ticipates in the reaction through a pushpull mechanism in which a hydride ion from NADPH is added to the carbonyl carbon of the aldehyde substrate while a proton from the imidazole ring of histidine is added to the carbonyl oxygen. The evidence in favor of this model is summarized as follows. Protection by NADPH and its analogue, AMP, against inactivation of the enzyme by phenylglyoxal implicated arginine in NADPH binding. The evidence for the involvement of lysine in the binding of 16oxohexadecanoate was supplied by the inactivation of the enzyme by pyridoxal-5’phosphate and the drastic increase in the Km for ~0x0 CIS acid as pH increased from 9 to 10. The postulate that the binding site for ~0x0 acid is a hydrophobic crevice with lysine at the bottom is supported by the effect of chain length on the K,,, for substrates. Complete inactivation of the enzyme by diethylpyrocarbonate and almost complete protection by NADPH but not by AMP suggested a catalytic role for histidine as proposed for lactate dehydrogenase. The molecular architecture and the catalytic mechanism of this dehydrogenase have much in common with those of other dehydrogenases. It appears that during evolution of this enzyme, the structural features essential for catalysis were preserved and the substrate binding site, namely the hydrophobic crevice with the lysine residue at the bottom, was acquired. REFERENCES

Plant Physiol. 59,667-672. V. P., AND KOLATTUKUDY,

P. E. (1978)

Arch. Biochem. Biophys., 191,452-465. 4. SWANN,

S., OEHLER,

R., AND BUSWELL,

R. (1943)

Org, Syntheses Coil. 2,276-277. 5. BROWN,

M. C., HEIM,

P., AND YOON,

J. Amer. Chem. Sot. 92,1637-X46. 6. COREY,

I?. J., AND SUGGS, J. W. (1975)

Tetrcrhed-

ron Lett. 2647. 7. CLELAND, W. W. (1970) in The Enzymes (Bover, P. D., id.), Vol. 2, pp. l-65, Academic. Pries.; New York. 8. EISENTHAL, R., AND CORNISH-BOWDEN, A. (1974) Biochem. J. 139, 715-720. 9. DAJZIEL, K. (1975) in The Enzymes (Boyer, P. D., ed.), Vol 11, Part A, pp. 2-52, Academic Press, New York. 10. ZEWE, V., AND FROMM, H. J. (1962) J. Biol. Chem. 237,1668-1675. 11. WRATTEN, C. C., AND CLELAND, W. W. (1963)

Biochemistry

2,935-941.

12. Hsu, R. Y., LARDY, H. A., AND CLELAND, W. W. (1967) J. Biol. Chem. 242.5315-5322. 13. RAVAL, D. N., AND WOLFE, R. G. (1962) Biochemistry 1, 1112-1117. 14. FROMM, H. J., AND NELSON, D. R. (1962) J. Biol

Chem. 237,215-220. 15. ORSI, B. A., AND CLELAND, W. W. (1972) Biochemistry l&102-109. 16. NETTER, H. (1969) Theoretical Biochemistry, p. 638, Oliver and Boyd, Ltd., Great Britain. 17. FRIEDEN, C. (1959) J. Bid. Chem. 234, X19-814. 18. LANGE, L. G., RIOFLDAN, J. F., AND VALLEE, B. L. (1974) Biochemistry 13,4361-4370. 19. YANG, P. C., AND SCHWERT, G. W. (1972) Biochemistry 11,2218-2224. 20. BERGHAUSER, J., AND FALDERBAUM, I. (1971)

Hoppe-Styler 21. TAKAHASHI, K. 6171-6179. 22. OVADI, J., LIBOR,

2. Physiol. Chem. 352.1189. (1968) J. Biol. Chem. 243,

S., AND EL~~DI, P. (1967) Arch. Biochem. Biophys. Acad. Sci. Hung. 2,455. 23. WINER, A., AND SCHWERT, G. (1959) J. Biol. Chem. 234.1155-1161. 24. MORRIS, D. L., AND MCKINLEY-MCKEE, (1972) Eur. J. B&hem. 29,515-520. 25. BLUMENTHAL, K., AND SMITH, E. (1973)

J. S. J.

Biol.

Chem. 248,6002-6008. 26. MCKINLEYMCKEE, J. S., AND MORRIS, D. L. (1972) Eur. J. B&hem. 28, l-11. 27. KOLATTUKUDY, P. E., AND AGRAWAL, V. P. (1974)

Lipids 9,662-691.

1. KOLATTUKUDY, P. E. (1978) in Biochemistry of Wounded Plant Storage Tissues (Kahl, G., ed.), Walter De Gruyter & Co., New York, in preen. 2. AGRAWAL, V. P., AND KOLATTUKUDY, P. E. (1977) 3. AGRAWAL,

KOLATTUKUDY

N. M. (1970)

28. KOLATTUKUDY, P. E., KRONMAN, K., AND PouLOSE, A, J. (1975) Plant Physiol. 55, 567-573. 29. DAVIES, D. D., PATIL, K. D., UGOCHUKWU, E. N., AND TOWERS, G. H. N. (1973) Phytochemistry 12, 523-530. 30. DAVIES, D. D., UGOCHUKWU, E. N., PATIL, K. D., AND TOWERS, G. H. N. (1973) Phytochemistry 12,531-536. 31. CLELAND, W. W. (1963) B&him. Biophys. Acta 67.104-137.

Mechanism of action of a wound-induced omega-hydroxyfatty acid:NADP oxidoreductase isolated from potato tubers (Solanum tuberosum L).

ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 2, December, AND BIOPHYSICS pp. 466-478, 1978 Mechanism of Action of a Wound-Induced w-Hydroxyfatty Acid:NADP...
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