Journal of Protein Chemistry, Vol. 11, No. 1, 1992

Modification of Aldehyde Dehydrogenase with Dicyclohexylcarbodiimide: Separation of Dehydrogenase from Esterase Activity Darryl P. Abriola I and Regina Pietruszko 2

Received August 19, 1991

Dehydrogenase activity of the cytoplasmic (El) isozyme of human liver aldehyde dehydrogenase (EC 1.2A.3) was almost totally abolished (3% activity remaining) by preincubation with dicyclohexylcarbodiimide(DCC), while esterase activity with p-nitrophenyl acetate as substrate remained intact. The esterase reaction of the modified enzyme exhibited a hysteretic burst prior to achieving steady-state velocity; addition of NAD + abolished the burst. The K,, for p-nitrophenyl acetate was increased, but physicochemicalproperties remained unchanged. The selective inactivation of dehydrogenaseactivity was the result of covalent bond formation. Protection by NAD + and chloral, saturation kinetics, and the stoichiometry and specificity of interaction indicated that the reaction of DCC occurred at the active site of the E 1 isozyme. The results suggested that some amino acid other than aspartate or glutamate, possibly a cysteine residue, located on a large tryptic peptide of the E1 enzyme, may have reacted with DCC. KEY WORDS: Aldehyde dehydrogenase; dicyclohexylcarbodiimide; chemical modification.

1. INTRODUCTION

Eckfeldt and Yonetani, 1976; Vallari and Pietruszko, 1981).

Aldehyde dehydrogenase (EC 1.2.1.3) catalyzes dehydrogenation of a large variety of aldehydes as well as hydrolysis of esters. Human aldehyde dehydrogenase consists of three isozymes: El, E2, and E3. The E1 (cytoplasmic) and E2 (mitochondrial) isozymes have been available homogeneous since 1977 (Greenfield and Pietruszko, 1977), while the E3 isozyme has been purified recently (Kurys et al., 1989). The primary structures of all three isozymes are now available (Hempel et al., 1984, 1985; Hsu et al., 1985; Kurys et al., 1991). While NAD + functions as an obligatory second substrate for dehydrogenase reaction, it is not essential for the esterase reaction. However, both NAD ÷ and NADH stimulate the esterase reaction (Feldman and Weiner, 1972; Sidhu and Blair, 1975;

Dehydrogenase Reaction Aldehyde + NAD + + H 2 0 . 4 Acid + NADH + H + Esterase Reaction Ester + H20 --* Alcohol + Acid Both reactions are irreversible, and proceed via a covalent intermediate, which, up to the present time, was thought to be the same for both reactions. However, there have been reports claiming that aldehyde dehydrogenation and esterase hydrolysis occur at different sites (Kitson, 1978; MacGibbon et al., 1978; Deady et al., 1985; Tu and Weiner, 1988a, b). The esterase and dehydrogenase activities of aldehyde dehydrogenase, however, have not been separated until this time. Carbodiimides have been used routinely for the synthesis of esters and amides, including peptides. Their general mode of action is via "activation" of a

t Center of Alcohol Studies, Rutgers University, Piscataway, New Jersey 08855-0969. 2 To whom all correspondence should be addressed.

59 0277-8033/92/0200-0059506.50/0© 1992PlenumPuNishingCorporation

60

Abriola and Pietruszko

© © N

II c

*NH II C ~ II N

H 4L i

II

N

NH I C* II N

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O-ocylisourea

d © NH I C=O I NH

d ~O E-C,~O.

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NH

d

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E- C ~ O N-R

Fig. 1. Reaction of DCC with protein carboxylgroups. DCC activates carboxyl groups to form an intermediateO-acylisourea,which can then react in one of several ways. If an excessof some nucleophilic species is added (right pathway), a stable enzyme-acylnucleophilecan result, liberatinga urea product. Wherewater is the displacing nucleophile(left pathway), the carboxylgroup originally derivatized is simply regenerated. Finally, under some conditions, an intramolecularacyl transfer can be favored, where the O-acylisourea rearranges to an N-acylurea (center pathway). In a special case of the pathway on the right, "R" can be a group on the protein itself (such as a nearby lysine side chain), or on another protein, producing a cross-linked protein.

carboxyl group to yield an intermediate O-acylurea which is then susceptible to attack by a nucleophilic group such as an amine, thiol, carboxyl, etc. In the area of protein modification, carbodiimides have probably been most useful in the estimation o f the total number ofcarboxyl groups present in proteins (Hoare and Koshland, 1967). To a more limited extent, carbodiimides have also beehluSeful in the chemical modification of specific amino acid residues. In this regard they have been used to identify active site residues of certain enzymes and in other structure-function studies. Reactions of carbodiimides with proteins are usually via derivatization of carboxyl groups of amino acids. In addition, however, carbodiimides have been found on occasion to react with the hydroxyl groups of serine or tyrosine or with the sulfhydryl group of cysteine (Carraway and Koshland, 1968; Carraway and Triplett, 1970). Amino groups can also react directly with carbodiimides (Takata et al., 1985), but are more commonly involved as the displacing nucleophile in a reaction initiated with a carboxyl group. In reacting with protein carboxyl groups, carbodiimides cause formation of an acyl urea, which can then react in one of three different ways (Fig. 1). In the first possible scheme, water can displace the reagent, regenerating the carboxyi group on the protein and forming a urea product. Alternatively, if some nucleophile is added in large excess (glycine methyl ester, glycine ethyl ester, glycinamide, methylamine, and aniline are all commonly used), it may be possible to displace the reagent and form a stable amide bond in its place. Finally, in some cases a rearrangement reaction is more facile than the other two possible routes, for reasons which may relate to the protein environment, reagent structure, or other steric factors. In this case, a somewhat more stable N-acylisourea is generated from the O-acyl species. One additional possible reaction is actually a specific example of displacement by a nucleophile; this is a cross-linking reaction, which can occur by reaction of an amino group (either a terminal amino group or the side chain of a lysine residue). Cross-linking can either involve a group on the same protein molecule or on a different molecule. When a radioactively labeled carbodiimide is employed in modification of a carboxyl group and displacement by a nucleophilic group occurs (including displacement by protein amino groups in crosslinking), the resulting protein loses the label. 2. MATERIALS AND M E T H O D S 2.1. Materials

N,N'-Dicyclohexylcarbodiimide (DCC) and aniline were from Aldrich. Unlabeled glycine ethyl ester,

Human Aldehyde Dehydrogenase sodium dodecyl sulfate, trichloroacetic acid, ammonium bicarbonate, and protein or peptide standards were from Sigma. N,N-Dicyclohexyl-[~4C] carbodiimide ([14C]-DCC) and [ring-G-3H] aniline ([3Hi-aniline) were from Amersham, and [glycine-1~4C] glycine ethyl ester hydrochloride ([14C]-glycine ethyl ester) and Biofluor scintillation cocktail were from New England Nuclear. TPCK-treated trypsin and N A D + were from Boehringer Mannheim, Inc. Guanidine hydrochloride was of the highest grade available from Sigma or US Biochemical. lodoacetic acid from Sigma was recrystallized from petroleum ether before use. 2-Mercaptoethanol was from Sigma or Aldrich. Organic solvents for reversed-phase HPLC (methanol and acetonitrile) were HPLCgrade, from Fisher or Aldrich. Triethylamine, acetic acid, and trifluoroacetic acid were all of the highest grade available from Pierce. All other materials were of reagent grade.

2.2. Enzyme Preparation The E1 isozyme of human liver aldehyde dehydrogenase was purified to homogeneity following the procedure of Hempel et al. (1982). Homogeneity was confirmed by isoelectric focusing, and specific activity. The enzyme was stored at 4°C in nitrogenated 30 mM sodium phosphate, pH 6.0, containing 1 mM EDTA, N A D + (1 mg/ml), and 0.1% (v/v) 2-mercaptoethanol. Prior to use, the enzyme was dialyzed in a Schleicher and Schuell apparatus with a collodion membrane, against eight changes of nitrogen-saturated 30 mM sodium phosphate, pH 6.0, containing 1 mM EDTA, to remove NAD + and mercaptoethanol.

61 coefficient of 9.8 raM- I cm- I was employed in calculations. Concentrations of stock solutions of the ester were determined in 0.1 N sodium hydroxide solution, using an extinction, coefficient of 18 mM -~ cm -I (Kezdy and Bender, 1962). Protein concentrations were determined by both 280 nm absorption (Greenfield and Pietruszko, 1977) and the procedure of Lowry et aL (1951) with bovine serum albumin as a standard.

2.4. Inactivation of the E1 Isozyme with DCC Inactivation of the E1 isozyme was carried out by incubating the enzme (1.0-10.0/~M final concentration) with DCC (10-200/zM final concentration) in 30 or 50 mM sodium phosphate buffer (pH 6.0 or 7.0, with 1 mM EDTA, degassed and nitrogenated at room temperature) at 25°C for several hours, except where stated otherwise. Stock solutions of DCC were prepared in either 95% ethanol or absolute ethanol; aliquots of these stocks were diluted in buffer to half the final incubation volume. The enzyme was then diluted to half the final volume, and the halves mixed to start the incubation. The final concentration of ethanol in the incubation was kept to 2% (v/v) or less. Inactivation of the enzyme was followed by the dehydrogenase and esterase activity assays described above. Where [14C]-DCC was employed, the radiolabeled material was supplied in toluene; toluene was evaporated under a stream of nitrogen, and an unlabeled stock of DCC was then added to the residue to obtain radiolabeled material of lower specific activity. Specific activity was determined by subjecting an aliquot of the final solution to scintillation counting on an LKBI219 Rackbeta liquid scintillation counter.

2.3. Enzyme Assay Included in the standard dehydrogenase assay mixture were 0.1 M sodium pyrophosphate, pH 9.0, 500/~M NAD +, 1 mM propionaldehyde, and 1 mM EDTA in a 3.0 mL total volume. Reactions were initiated by the addition of enzyme, and time progress curves were recorded on a Varian 635 recording spectrophotometer or a Gilford 252 updated Beckman DU spectrophotometer at 25°C in 1-cm light path cuvettes by following the production of N A D H at 340 nm and by using an extinction coefficient of 6.22mM -~ cm -~. Esterase activity was measured using 150/zM p-nitrophenyl acetate as substrate, in 50 or 100 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA. Production of p-nitrophenolate ion was monitored at 400 nm, and an extinction

2.5. Determination of Stoichioraetry of Modification Following modification with [IaC]-DCC,the protein was separated from free reagent either by precipitation with ice-cold 10% (v/v) trichtoroacetic acid or by gel filtration through Sephadex G-50 fine (1.7 x 18 cm) equilibrated with 30 mM or 50 mM sodium phosphate buffer (pH 6.0 or 7.0, with 1.0 mM EDTA). When precipitated, the enzyme was centrifuged (l 1,000-50,000 g for 5-20 rain) and the precipitate was washed three times with distilled water. The E1-DCC adduct was redissolved in 6.0 M guanidine hydrochloride/0.1 M Tris-HCl, pH 6.0--8.0, with 2.0 mM EDTA, and absorbance at 280 nm vs. guanidine hydrochloride buffer alone used to calculate protein concentration. The bound reagent did not

62

Abriola and Pietruszko

contribute to absorbance in this region. When denaturation of the modified protein was not carried out, absorbance at 280 nm vs. the buffer alone was determined. An aliquot of the solution was then subjected to scintillation counting to determine the amount of reagent bound.

for scintillation counting. Following stoichiometry determinations, the sample was thoroughly nitrogenated under a stream of nitrogen and sealed for storage at 4°C. At 40 hr and 6 days, aliquots were passed through a gel filtration column and the enzyme activity and stoichiometry were calculated.

2.6.2. After Denaturation

2.6. Stability of EI-DCC Adduet

2.6.1. Before Denaturation Incubations of the E1 isozyme with either [14C]D C C in ethanol or ethanol alone were carried out at 25°C for 36 hr, in degassed and nitrogenated 50 m M sodium phosphate, p H 7.0, containing 1 m M EDTA. The reaction mixtures were passed through a gel filtration column o f Sephadex G-50 fine (1.7 x 18 cm) equilibrated with 50 m M sodium phosphate buffer, p H 7.0. The protein was calculated by determining absorbance at 280 nm, and an aliquot was also used

The enzyme was denatured by adding trichloroacetic acid. Following centrifugation, the protein pellet was washed three times with ice-cold distilled water, centrifuging and discarding the supernatants after each wash. Finally, the pellet was dissolved in 6 . 0 M guanidine hydrochloride/0.1 M Tris-HC1/ 2 m M EDTA, p H 6.0, and absorbance at 280 nm was determined vs. guanidine alone. In addition, an aliquot was taken for scintillation counting to determine the reagent bound. A portion of this solution of denatured modified enzyme was put into each of three

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Fig. 2. Effectsof preincubation of El enzyme with DCC on final steady-state esterase and dehydrogenase activities. E! enzyme (4.4 pM final concentration) was incubated with 150 (I-1, esterase, and A, dehydrogenase, activity) or 200 ( I , esterase, and A, dehydrogenase, activity) #M DCC in 50 mM sodium phosphate buffer,pH 7.0, containing l mM EDTA, at 25°C. Dehydrogenaseactivity was monitored by withdrawing aliquots of the incubation mixture at various time intervals and measuring activity with propionaldehyde and NAD ÷ in the standard assay system. Steady-state esterase activity was measured at pH 7.0, in 50 mM sodium phosphate buffer, with 150pM pnitrophenyl acetate, and calculated (as described in Methods) after the initial hysteretic burst was concluded (~30 min after start of assay).

Human Aldehyde Dehydrogenase collodion bags for dialysis vs. buffers of different pH. Dialysis was then carried out over a period of 40 hr, changing the buffer at intervals (six in all) and determining the amount of radioactivity in each change of buffer. Based upon the specific activity of the reagent used, rates of reagent loss were calculated. At the end of dialysis, the modified protein remaining was dissolved in guanidine hydrochloride (6.0 M) for measurement of protein and scintillation counting. A similar procedure was followed for dialysis of the denatured E1-DCC adduct into 70% formic acid. 2.7. Preparation of Trypsin-Digested DCC-Modified Enzyme and HPLC Following incubation, the enzyme was precipitated with trichioroacetic acid, washed, and equilibrated in ammonium bicarbonate buffer to bring it directly into conditions for tryptic digestion. Carboxymethylation and digestion with TPCK-trypsin were as described previously (Hempel and Pietruszko, 1981). After tryptic digestion, the modified enzyme was either directly subjected to column chromatography, or lyophilized and redissolved in 0.1% (v/v) trifluoroacetic acid, or 0.1 M triethylamine acetate buffer for gel filtration or reversed-phase HPLC. Reversed-phase H P L C was done employing a C-18silica column with a gradient system of 0.1% TFA to 100% methanol over 80 rain with a flow rate of 1.0-1.5 ml/min. 3. RESULTS 3.1. Effect of DCC on Dehydrogenase and Esterase Activities Preincubation of the cytoplasmic (El) isozyme with DCC caused loss of the dehydrogenase activity (Fig. 2). The activity loss was initially rapid; at approximately 10-15% activity remaining the activity loss slowed down considerably. Incubation of the enzyme with 2 0 0 p M DCC for a total of 36hr decreased the activity to 3% activity remaining. The enzyme retained esterase activity (Fig. 2). Loss of dehydrogenase activity of the El isozyme was the same when measured with both 1.0 and 10.0 mM propionaldehyde and thus was not due to an increase in the Km for propionaldehyde. 3.2. Esterase Activity of the Modified Enzyme The properties of the esterase reaction catalyzed by the modified enzyme were different from those

63

2.0.