Biochimica et Biophysica Acta, 1! 19 (1992) 69-73 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
69
BBAPRO 34097
Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase Kirill M. Popov, Natalia Y. Kedishvili and Robert A. Harris Department of Biochemistn, and Molecular Biology, Indiana Unicersity School of Medicine, h~dianapolis, IN (U.S.A.)
Key words: Methylmalonate semiaidehyde dehydrogenase, Methylmalonate semialdehyde; Malonate semialdehyde: p-Nitrophenyl acetate: Esterase, Aldehyde dehydrogenase: Acetyl-CoA
Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD ÷ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyi groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyI-CoA. These findings suggest that an $-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction. Introduction Methylmalonate semialdehyde dehydrogenase (EC 1.2.1.27) (MMSDH) catalyzes the irreversible oxidative decarboxylation of malonate and methylmalonate semialdehydes with concomitant reduction of NAD + and synthesis of acetyl- and propionyl-CoA, respectively [1,2]. Purification of the mammalian enzyme and characterization of some of its physical and kinetic properties were described in the previous paper from this laboratory [3]; however, the catalytic mechanism of the enzyme has not been investigated. A common mechanism of catalysis has been suggested for several NAD+-dependent dehydrogenases [4]. This mechanism is based on the high sensitivity of such enzymes to sulfhydryl reagents and on the ability of the sulfhydryl groups of these enzymes to readily produce thiohemiacetals. According to this hypothesis, interaction between the aldehyde substrate and an active site sulfhydryl group of the enzyme leads to formation of a thiohemiacetal, which is then oxidized to a thioester with concomitant reduction of NAD +.
Abbreviations: MMSDH, methyimalonate semialdehyde dehydrogenase; ALDH, aldehyde dehydrogenase; GAPDH, glyceraldehyde-3phosphate dehydrogenase; D'VI', dithiothreitol: NEM, N-ethyimaleimide; CoA, coenzyme A, Pi, inorganic phosphate. Correspondence: R.A. Harris, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202-5122, U.S.A.
The thioester can be hydrolyzed with water to give the acid as the reaction product in the case of aldehyde dehydrogenase (ALDH) [5] or can be subject to phosphorolysis by inorganic phosphate (Pi) to give a mixed anhydride in the case of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [6]. Since the active site sulfhydryl groups of these enzymes would be expected to be strong nucleophiles, the hypothesis was proposed that these aldehyde dehydrogenases should also catalyze an esterase reaction [7], e.g., the hydrolysis of p-nitrophenyl acetate. Indeed, this catalytic property has been demonstrated for both GAPDH [7] and ALDH [6,8]. The methylmalonate semialdehyde dehydrogenase is of special interest because of its unique ability to form CoA esters in a reaction coupled to its aldehyde dehydroger~ase activity. Whether its reaction mechanism is basically the same as that of aldehyde dehydrogenases that simply convert aldehydes to carboxylic acids without CoA ester formation remains to be determined. There are some indications already [I,3] that a highly reactive sulhydryl group is essential for the reaction catalyzed by MMSDH. Since the same sulfhydryl group may also function as an esterase, the possibility was investigated that MMSDH would hydrolyze pnitrophenyl acetate. This paper reports on the properties of the esterase activity discovered for MMSDH and the implications of the findings with respect to the probable intermediate common to the esterase reaction and the dehydrogenase/CoA ester synthetic reaction.
70
l
Experimental procedures
20
Materials. NAD +, NADH and p-nitrophenyi acetate were purchased from Sigma. Ethyl 3,3-diethoxypropionate was obtained from Aldrich and converted to the potassium salt of malonate semialdehyde as described previously [3]. Sources of other chemicals were as described previously [3], Preparation of MMSDH. Rat liver MMSDH was prepared and stored as described previously [3] with modifications to be described elsewhere (Kedishvili, N.Y., Popov, K.M. and Harris R.A., unpublished data). Preparation of apo-enzyme. MMSDH at a protein concentration of 1-2 m g / m l and suspended in a buffer 10 mM in Tris-HC! (pH 8.0), 0.1 mM in EDTA, 1 mM in D T r , 0.5 mM in NAD + and 2 M in ( N H 4 ) 2 S O 4 w a s incubated for 10-15 min at room temperature and then desalted on a Sephadex G-25 fast-flow column equilibrated with 15 mM potassium phosphate (pH 7.8) and 0.1 mM EDTA. This procedure has been established to efficiently remove NAD ÷ from the enzyme (less than 0.01 tool NAD + remained per tool of the enzyme subunits)(Kedishvili, N.Y., Popov, K.M. and Harris, R.A., unpublished data). This preparation was used directly for determination of esterase and dehydrogenase activities. Assay of enzyme activity. In order to facilitate the direct comparison of the esterase and dehydrogenase reaction rates, both activities were determined in 50 mM potassium phosphate (pH 7.8) and 0.1 mM EDTA buffer at 30°C. Standard solutions of p-nitrophenyl acetate were prepared with acetone as solvent to minimize spontaneous hydrolysis. Acetone did not affect enzyme activity provided its concentration was less than 2% in the assay solution. Dehydrogenase activity was measured by following NADH production at 340 nm as described previously [3]. Esterase activity was assayed by following p-nitrophenol production at 400 nm ( E = 16 x 10 a M -I cm-J). Corrections were made for spontaneous hydrolysis• Acetyl-CoA determination. To determine the esterase reaction product produced in the presence of CoA, 1 ml of a solution containing. 0.3-0.5 mg of MMSDH and 250 nmol p-nitrophenyl acetate was incubated at 30°C until the reaction reached steady state and the CoA or NADH plus CoA were added. The reaction was allowed to proceed until 100 nmol of p-nitrophenyl acetate had been hydrolyzed. AcetyI-CoA was determined in perchloric acid extracts neutralized with KOH by the method of Decker [9].
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Fig. 1. Spectrophotomeric measurement of the formation of an acyi enzyme complex. The reaction mixture (1 ml) contained 250 ttM p-nitrophenyi acetate and 17 (o) or 13 (e) nmol of MMSDH subunits per ml. Lower curve, data obtained when 17 nmol of MMSDH subunits were preincubated with 100-fold molar excess of NEM for 1 rain. The reaction was started with p-nitrophenyl acetate.
etate (Fig. l). The time-course revealed a burst of p-nitrophenol production during the first 2 min, followed by a slow but continuous formation of this product, suggesting that the hydrolysis of an intermediate (presumably an S-acyl enzyme) is a rate-limiting step of the esterase reaction. Both the extent of the burst and the steady state formation rate were proportional to the amount of the enzyme added• For the steady state phase, K m and Vm~ values of 12 + 2/.tM and 0.7 _ 0.2 nmol/min per mg protein (mean _+S.D. for four preparations), respectively, were determined. Based on the amount of enzyme subunits present in the reaction mixture, it can be calculated from the extent of the initial burst that 0.9 mol of acetyl groups were bound per tool of MMSDH subunits. Values of 0.85 to 0.95 were obtained in a number of other experiments (0.90 _+ 0.05, mean _+S.D. for four preparations of MMSDH), and this ratio was independent of the protein concentration• Preincubation of the enzyme with N-ethylmaleimide (NEM) to irreversibly modify sulfhydryl groups of the protein, inhibited the formation of the acyl-enzyme complex and also the ability of the enzyme to catalyze the hydrolysis of p-nitrophenyl acetate. This provides evidence that reactive sulfhydryl groups are essential for p-nitrophenyl acetate hydrolysis and is consistent with the enzyme intermediate being a thioester.
Results Time-course of p-nitrophenyl acetate hydrolysis by MMSDH Highly purified preparations of rat liver MMSDH exhibited esterolytic activity toward p-nitrophenyl ac-
Effects of NAD + and NADH on esterolytic activity of MMSDH The effect of NAD + was examined over the concentration range of 0 to 500/.tM at a fixed p-nitrophenyl acetate concentration of 250/.~M. NAD + at the high-
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Fig. 2. Activation of esterase activity by NAD +. Reaction mixture (1 mi) contained 16 nmol of MMSDH subunits, 250 /.tM of pnitrophenyl acetate and indicated concentrations of NAD +. Insert: double reciprocal plot of rate of accelerated p-nitrophenyl acetate hydrolysis versus NAD + concentration. Vo refers to basal steady state esterase activity in the absence of NAD*. V refers to esterase activity in the presence of NAD +.
est concentration tested (500 # M ) increased the apparent Vmax by a factor of 2 (Fig. 2). Plotting the reciprocal of the increase in the hydrolysis rate vs. the reciprocal concentration of N A D + revealed a K D of 110 + 10 # M (Fig. 2, insert). The velocity of the esterase reaction was investigated in the presence of various concentrations of NADH (Fig. 3). In a manner analogous to N A D +, NADH increased the rate of hydrolysis of p-nitrophenyl acetate. A K D of 10.0+0.5 # M for N A D H was calculated from double reciprocal plots. In
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Fig. 4. Activation of esterase activity by CoA and pyridine nucleotides. Reaction mixture (1 ml) contained 12 nmol of MMSDH subunits, 250/~M of p-nitrophenyl acetate, different concentrations of CoA and, ~vhere indicated either NAD + (100/zM) or NADH (100 /.tM). El, no pyridine nucleotide; *, NAD+; o, NADH. All activities were corrected for basal activities corresponding to the three different incubation conditions. Basal activities were 0.4 nmol/min in the absence of pyridine nucleotides, 0.75 nmol/min in the presence of NAD + and 6.4 nmol/min in the presence of NADH. Insert: double reciprocal plots of the rate of accelerated p-nitrophenyi acetate hydrolysis versus the CoA concentration under the different reaction conditions. V-V0 refers to velocity of the reaction corrected for the basal activities given above.
contrast to NAD +, the extent of the stimulatory effect of N A D H on the rate of esterase reaction was greater, reaching 16-fold at saturating concentrations of the reduced pyridine nucleotide.
Effect of CoA on the rate of p-nitrophenyl acetate hydrolysis by MMSDH
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Fig. 3. Activation of e~erase activity by N ~ H . Reaction mixture (1 ml) contained 12 nm~ of MMSDH subunits, 250 /LM of pnitrophenyl acetate and indicated concentrations of NADH. Insert: double reciprocal plot of rate of accelerated p-nitrophenyl acetate hydrolysis versus N A D H concentration. Vo ~efers to basal esterase activity in the absence of N A D H . I/refers to esterase activity in the presence of NADH.
CoA increased the rate of p-nitrophenyl acetate hydrolysis by a factor of 7 (Fig. 4). An apparent activation constant of 60 + 4 # M was determined from double reciprocal plots (Fig. 4, insert). In the presence of 100 /.~M NADH, the stimulating effect of CoA was 16-17-fold and the apparent activation constant decreased to 29 + 3 /~M. The extent of increase in the esterase reaction rate approached 32-fold in the presence of both effectors (basal activity of 0.4 nmol/min in the absence of CoA and NAD +, activity of 12.8 nmol/min in the presence of CoA and NAD+); a rate close to the acetylation reaction measured from the initial burst data. NAD +, in contrast to NADH, was practically without effect on the ability of CoA to deacetylate MMSDH (Fig. 4). Moreover, CoA had some inhibitory effect on the ability of NAD + to stimulate the hydrolysis of p-nitrophenyl acetate.
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TABLE I Synthesis of acetyI-CoA from p-nitrophenyl acetate a Additions
p-Nitrophenol released (nmol)
AcetyI-CoA synthesized (nmol)
CoA CoA + NADH
91 _+4 86 +_3
95 +_ 8 75 _+10
MALONATE
SEMIALDEHYOE
/
" The products of the esterase reaction were determined in a ~eaction mixture (1 ml) (containing 0.3-0.5 mg MMSDH, 250 nmol p-nitrophenyi acetate, 100 nmol CoA and, where indicated, 100 nmol NADH as described in Materials and Methods. Values are the mean _-!:.S.E. for three determinations.
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TIME, MIN. Fig. 5. Inhibition of esterase activity by malonate semialdehyde. Assay conditions were essentially as in Fig. 4. NAD ÷, (100 /zM), CoA (100/~M) and malonate semialdehyde (1 mM) were added as indicated by the arrows.
Malonate semialdehyde inhibits the esterase reaction and p-nitrophenyl acetate inhibits the dehydrogenase reaction Malonate semialdehyde (1 mM), a substrate for the dehydrogenase activity of the enzyme, effectively inhibited ester hydrolysis in the presence of NAD ÷ and CoA. Fig. 5 shows the pattern of malonate semialdehyde inhibition at a concentration of NAD + close to its activation constant (100 izM) and a CoA concentration of 100/.tM. p-Nitrophenyl acetate efficiently inhibited the dehydrogenase reaction catalyzed by MMSDH. The inhibitory constant for 50% inhibition (IC50) was 150 +_ 7 100
Determhzation of the product of the esterase reaction proceeding in the presence of CoA Acetyl-CoA was formed as a product when MMSDH is incubated with p-nitrophenyl acetate and CoA (Table I). The amount of acetyl-CoA synthesized was nearly equal to the amount of p-nitrophenol released. These data indicate that the acyl intermediate formed from p-nitrophenyl acetate is subject of transacetylation in the presence of CoA. NADH, shown above to be an activator of the esterase reaction, did not interfere with the synthesis of acetyl-CoA (Table I), Discussion
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50
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/~M measured with 50 tiM malonate semialdehyde as substrate (Fig. 6). In separate experiments, pnitrophenyl acetate was found to be a competetive inhibitor of the dehydrogenase reaction with K i = 17 _+ 3 /zM (data not shown). The competitive nature of inhibition by p-nitrophenyl acetate together with inhibition of the esterase reaction by malonate semialdehyde provide evidence that p-nitrophenyl acetate is hydrolyzed at the semialdehyde binding site.
1000
p-NWROPHENYL ACETATE, FM Fig. 6. Inhibition of dehydrogenase activity of MMSDH by pnitrophenyl acetate. MMSDH activity was measured in the presence of 50/~M malonate semialdehyde, 300/~M CoA and 0.5 mM NAD + Specific activity of MMSDH was 7.8 U / r a g protein and 0.5 # g of enzyme protein was used in each assay (1 ml). Each point represents mean + S.D. of four determinations.
The ability of rat liver MMSDH to catalyze ester hydrolysis as well as semialdehyde oxidation has been documented in the present paper. The findings that hydrolysis of p-nitrophenyl acetate by MMSIDH was inhibited by malonate semialdehyde, a substrate of dehydrogenase reaction, and conversely that the dehydrogenase reaction was inhibited by p-nitrophenyl acetate provide evidence for ester hydrolysis taking place at the catalytic center for dehydrogenase activity. This conclusion is also in a good agreement with the finding of regulation of esterolytic activity by pyridine nucleotides (Figs. 2 and 3) and CoA (Fig. 4). The activation constant values for NAD + (110 ~M), NADH (10 /zM) and CoA (29 tzM), determined for the esterase reaction, were reasonably close t o K m values for NAD ÷ (150 t~M) and CoA (30 #M) and the K i value for NADH (3.1 /zM) determined for the dehydrogenase reaction [3].
73 Some aspects of the esterase reaction catalyzed by MMSDH are similar to previous observations made for ALDH [6] and GAPDH [5]. The stimulatory effects of pyridine nucleotides on esterolytic activity of MMSDH were similar to their effects of ALDH, but the magnitude of these effects was different. In the case of ALDH both NAD + and NADH had exactly the same 2-fold stimulatory effect [6], whereas in the case of MMSDH, NADH was 8-times more potent than NAD +. In greater contrast, NAD + is a strong inhibitor of the esterolytic activity of GAPDH [5]. On the other hand, MMSDH and GAPDH exhibited very similar time courses for p-nitrophenyl acetate hydrolysis. With both enzymes, the reaction occurs with an initial burst of p-nitrophenol production, suggesting hydrolysis of an S-acyl enzyme as the rate-limiting step. In the case of MMSDH, CoA - the natural acceptor of acyl groups accelerated the esterase reaction by 7-fold. In the presence of NADH, the overall enhancement was about 32-fold, i.e. close to the acetylation rate measured from the initial burst of p-nitrophenol formation. However, it was not possible to force the esterase reaction to proceed at a rate faster than the dehydrogenase/CoA ester synthetic reaction. The fact that the stimulated esterase reaction was still slower suggests that the rate limiting step involves the acylation by p-nitrophenyl acetate rather than deacetylation of the enzyme-thioester. If deacetylation were rate limiting, the Vm~, for p-nitrophenyl ester hydrolysis would have been equal to or greater than that found for the dehydrogenase/CoA ester synthetic reaction. Thus, in the absence of CoA and NADH the breakdown of S-acetyl enzyme may be a rate limiting step, whereas in the presence of both effectors the rate limiting step occurs prior to the deacetylation step. The f'mding that rat liver MMSDH catalyzes the hydrolysis of p-nitrophenyl acetate strongly supports the hypothesis that an enzyme-thioester: O
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is an important intermediate in the reaction pathway. Whether the formation or breakdown of an S-acyl enzyme intermediate is the rate limiting step for the dehydrogenase reaction catalyzed by rat liver MMSDH has not been determined. Observations reported here for the esterase activity make it clear that the deacylation reaction is rate limiting in the absence of CoA. As was shown for the analogous enzyme from Pseudomonas aeruginosa [1], the dehydrogenase reaction can proceed in the absence of CoA, but extremely slowly. It is, however, impossible to determine the
rate-limiting step for the overall dehydrogenase reaction proceeding in the presence of CoA because the rate of the deacylation reaction in the presence of NADH and CoA can not be readily estimated. A pre-steady state kinetic investigation of both reactions will be required to unequivocally determine which steps are actually rate limiting. For MMSDH from rat liver [3] and for the analogous enzyme from Pseudomonas aeruginosa [1,2], it is known that the oxidative decarboxylation of malonate and methylmalonate semialdehydes depends on the presence of CoA. Hydrolysis of the acyl enzyme in the presence of NADH and NAD + suggests that acetate or propionate could be products of the reaction. However, this probably does not occur in vivo as long as sufficient CoA is available since the rate of hydrolysis is negligible relative to the total rate of the dehydrogenase reaction. The question of whether hydrolysis could play a significant role in valine disposal under conditions of limited CoA availability is of interest but will require additional investigation.
Acknowledgments We thank Paul Rougraff for providing 3-hydroxyisobutyrate dehydrogenase which was needed for the estimation of MMSDH activity during purification. This work was supported by grants from the U.S. Public Health Services (NIH DK 40441), Showalter Foundation and the Diabetes Research and Training Center of Indiana University School of Medicine (DK 20542). KMP and NYK are postdoctoral fellows of the Indiana Affiliate of the American Heart Association.
References 1 Sokatch, J.R., Sanders, L.E. and Marshall, V.P. (1968) J. Biol. Chem. 243, 2500-2506. 2 Bannerjee, D., Sanders, L.E. and Sokatch, J.R. (1970) J. Biol. Chem. 245, 1828-1835. 3 Goodwin, G.W., Rougraff, P.M., Davis, E.J. and Harris, R.A. (1989) J. Biol. Chem. 264, 14965-14971. 4 Jakoby, W.B. (1963) in The Enzymes (Boyer, P.B., ed.), Vol. 7, pp. 203-214, Academic Press, New York. 5 Feldman, R.I. and Weiner, H. (1972) J. Biol. Chem. 247, 267-272. 6 Harris, J.I., Meriwether, B.P. and Park, J.H. (1963) Nature 198, 154-157. 7 Park, J.H., Meriwether, B.P., Ciodfeld, P. and Cunningham, L.W. (1961) J. Biol. Chem. 236, 136-141. 8 Sidhu, R.S. and Blair, A.H. (1975) J. Biol. Chem. 250, 7894-7898. 9 Decker, K. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. 4, pp. 1988-1993, Academic Press, New York. 10 Hartley, B.S. and Kilby, B.A. (1054) Biochem. J. 56, 288-295. 1| Racker, E. (1955) Physiol. Rev. 35, 1-56.
69
BBAPRO 34097
Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase Kirill M. Popov, Natalia Y. Kedishvili and Robert A. Harris Department of Biochemistn, and Molecular Biology, Indiana Unicersity School of Medicine, h~dianapolis, IN (U.S.A.)
Key words: Methylmalonate semiaidehyde dehydrogenase, Methylmalonate semialdehyde; Malonate semialdehyde: p-Nitrophenyl acetate: Esterase, Aldehyde dehydrogenase: Acetyl-CoA
Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD ÷ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyi groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyI-CoA. These findings suggest that an $-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction. Introduction Methylmalonate semialdehyde dehydrogenase (EC 1.2.1.27) (MMSDH) catalyzes the irreversible oxidative decarboxylation of malonate and methylmalonate semialdehydes with concomitant reduction of NAD + and synthesis of acetyl- and propionyl-CoA, respectively [1,2]. Purification of the mammalian enzyme and characterization of some of its physical and kinetic properties were described in the previous paper from this laboratory [3]; however, the catalytic mechanism of the enzyme has not been investigated. A common mechanism of catalysis has been suggested for several NAD+-dependent dehydrogenases [4]. This mechanism is based on the high sensitivity of such enzymes to sulfhydryl reagents and on the ability of the sulfhydryl groups of these enzymes to readily produce thiohemiacetals. According to this hypothesis, interaction between the aldehyde substrate and an active site sulfhydryl group of the enzyme leads to formation of a thiohemiacetal, which is then oxidized to a thioester with concomitant reduction of NAD +.
Abbreviations: MMSDH, methyimalonate semialdehyde dehydrogenase; ALDH, aldehyde dehydrogenase; GAPDH, glyceraldehyde-3phosphate dehydrogenase; D'VI', dithiothreitol: NEM, N-ethyimaleimide; CoA, coenzyme A, Pi, inorganic phosphate. Correspondence: R.A. Harris, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202-5122, U.S.A.
The thioester can be hydrolyzed with water to give the acid as the reaction product in the case of aldehyde dehydrogenase (ALDH) [5] or can be subject to phosphorolysis by inorganic phosphate (Pi) to give a mixed anhydride in the case of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [6]. Since the active site sulfhydryl groups of these enzymes would be expected to be strong nucleophiles, the hypothesis was proposed that these aldehyde dehydrogenases should also catalyze an esterase reaction [7], e.g., the hydrolysis of p-nitrophenyl acetate. Indeed, this catalytic property has been demonstrated for both GAPDH [7] and ALDH [6,8]. The methylmalonate semialdehyde dehydrogenase is of special interest because of its unique ability to form CoA esters in a reaction coupled to its aldehyde dehydroger~ase activity. Whether its reaction mechanism is basically the same as that of aldehyde dehydrogenases that simply convert aldehydes to carboxylic acids without CoA ester formation remains to be determined. There are some indications already [I,3] that a highly reactive sulhydryl group is essential for the reaction catalyzed by MMSDH. Since the same sulfhydryl group may also function as an esterase, the possibility was investigated that MMSDH would hydrolyze pnitrophenyl acetate. This paper reports on the properties of the esterase activity discovered for MMSDH and the implications of the findings with respect to the probable intermediate common to the esterase reaction and the dehydrogenase/CoA ester synthetic reaction.
70
l
Experimental procedures
20
Materials. NAD +, NADH and p-nitrophenyi acetate were purchased from Sigma. Ethyl 3,3-diethoxypropionate was obtained from Aldrich and converted to the potassium salt of malonate semialdehyde as described previously [3]. Sources of other chemicals were as described previously [3], Preparation of MMSDH. Rat liver MMSDH was prepared and stored as described previously [3] with modifications to be described elsewhere (Kedishvili, N.Y., Popov, K.M. and Harris R.A., unpublished data). Preparation of apo-enzyme. MMSDH at a protein concentration of 1-2 m g / m l and suspended in a buffer 10 mM in Tris-HC! (pH 8.0), 0.1 mM in EDTA, 1 mM in D T r , 0.5 mM in NAD + and 2 M in ( N H 4 ) 2 S O 4 w a s incubated for 10-15 min at room temperature and then desalted on a Sephadex G-25 fast-flow column equilibrated with 15 mM potassium phosphate (pH 7.8) and 0.1 mM EDTA. This procedure has been established to efficiently remove NAD ÷ from the enzyme (less than 0.01 tool NAD + remained per tool of the enzyme subunits)(Kedishvili, N.Y., Popov, K.M. and Harris, R.A., unpublished data). This preparation was used directly for determination of esterase and dehydrogenase activities. Assay of enzyme activity. In order to facilitate the direct comparison of the esterase and dehydrogenase reaction rates, both activities were determined in 50 mM potassium phosphate (pH 7.8) and 0.1 mM EDTA buffer at 30°C. Standard solutions of p-nitrophenyl acetate were prepared with acetone as solvent to minimize spontaneous hydrolysis. Acetone did not affect enzyme activity provided its concentration was less than 2% in the assay solution. Dehydrogenase activity was measured by following NADH production at 340 nm as described previously [3]. Esterase activity was assayed by following p-nitrophenol production at 400 nm ( E = 16 x 10 a M -I cm-J). Corrections were made for spontaneous hydrolysis• Acetyl-CoA determination. To determine the esterase reaction product produced in the presence of CoA, 1 ml of a solution containing. 0.3-0.5 mg of MMSDH and 250 nmol p-nitrophenyl acetate was incubated at 30°C until the reaction reached steady state and the CoA or NADH plus CoA were added. The reaction was allowed to proceed until 100 nmol of p-nitrophenyl acetate had been hydrolyzed. AcetyI-CoA was determined in perchloric acid extracts neutralized with KOH by the method of Decker [9].
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Fig. 1. Spectrophotomeric measurement of the formation of an acyi enzyme complex. The reaction mixture (1 ml) contained 250 ttM p-nitrophenyi acetate and 17 (o) or 13 (e) nmol of MMSDH subunits per ml. Lower curve, data obtained when 17 nmol of MMSDH subunits were preincubated with 100-fold molar excess of NEM for 1 rain. The reaction was started with p-nitrophenyl acetate.
etate (Fig. l). The time-course revealed a burst of p-nitrophenol production during the first 2 min, followed by a slow but continuous formation of this product, suggesting that the hydrolysis of an intermediate (presumably an S-acyl enzyme) is a rate-limiting step of the esterase reaction. Both the extent of the burst and the steady state formation rate were proportional to the amount of the enzyme added• For the steady state phase, K m and Vm~ values of 12 + 2/.tM and 0.7 _ 0.2 nmol/min per mg protein (mean _+S.D. for four preparations), respectively, were determined. Based on the amount of enzyme subunits present in the reaction mixture, it can be calculated from the extent of the initial burst that 0.9 mol of acetyl groups were bound per tool of MMSDH subunits. Values of 0.85 to 0.95 were obtained in a number of other experiments (0.90 _+ 0.05, mean _+S.D. for four preparations of MMSDH), and this ratio was independent of the protein concentration• Preincubation of the enzyme with N-ethylmaleimide (NEM) to irreversibly modify sulfhydryl groups of the protein, inhibited the formation of the acyl-enzyme complex and also the ability of the enzyme to catalyze the hydrolysis of p-nitrophenyl acetate. This provides evidence that reactive sulfhydryl groups are essential for p-nitrophenyl acetate hydrolysis and is consistent with the enzyme intermediate being a thioester.
Results Time-course of p-nitrophenyl acetate hydrolysis by MMSDH Highly purified preparations of rat liver MMSDH exhibited esterolytic activity toward p-nitrophenyl ac-
Effects of NAD + and NADH on esterolytic activity of MMSDH The effect of NAD + was examined over the concentration range of 0 to 500/.tM at a fixed p-nitrophenyl acetate concentration of 250/.~M. NAD + at the high-
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Fig. 2. Activation of esterase activity by NAD +. Reaction mixture (1 mi) contained 16 nmol of MMSDH subunits, 250 /.tM of pnitrophenyl acetate and indicated concentrations of NAD +. Insert: double reciprocal plot of rate of accelerated p-nitrophenyl acetate hydrolysis versus NAD + concentration. Vo refers to basal steady state esterase activity in the absence of NAD*. V refers to esterase activity in the presence of NAD +.
est concentration tested (500 # M ) increased the apparent Vmax by a factor of 2 (Fig. 2). Plotting the reciprocal of the increase in the hydrolysis rate vs. the reciprocal concentration of N A D + revealed a K D of 110 + 10 # M (Fig. 2, insert). The velocity of the esterase reaction was investigated in the presence of various concentrations of NADH (Fig. 3). In a manner analogous to N A D +, NADH increased the rate of hydrolysis of p-nitrophenyl acetate. A K D of 10.0+0.5 # M for N A D H was calculated from double reciprocal plots. In
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COENZYME A, FM
Fig. 4. Activation of esterase activity by CoA and pyridine nucleotides. Reaction mixture (1 ml) contained 12 nmol of MMSDH subunits, 250/~M of p-nitrophenyl acetate, different concentrations of CoA and, ~vhere indicated either NAD + (100/zM) or NADH (100 /.tM). El, no pyridine nucleotide; *, NAD+; o, NADH. All activities were corrected for basal activities corresponding to the three different incubation conditions. Basal activities were 0.4 nmol/min in the absence of pyridine nucleotides, 0.75 nmol/min in the presence of NAD + and 6.4 nmol/min in the presence of NADH. Insert: double reciprocal plots of the rate of accelerated p-nitrophenyi acetate hydrolysis versus the CoA concentration under the different reaction conditions. V-V0 refers to velocity of the reaction corrected for the basal activities given above.
contrast to NAD +, the extent of the stimulatory effect of N A D H on the rate of esterase reaction was greater, reaching 16-fold at saturating concentrations of the reduced pyridine nucleotide.
Effect of CoA on the rate of p-nitrophenyl acetate hydrolysis by MMSDH
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I 100
Fig. 3. Activation of e~erase activity by N ~ H . Reaction mixture (1 ml) contained 12 nm~ of MMSDH subunits, 250 /LM of pnitrophenyl acetate and indicated concentrations of NADH. Insert: double reciprocal plot of rate of accelerated p-nitrophenyl acetate hydrolysis versus N A D H concentration. Vo ~efers to basal esterase activity in the absence of N A D H . I/refers to esterase activity in the presence of NADH.
CoA increased the rate of p-nitrophenyl acetate hydrolysis by a factor of 7 (Fig. 4). An apparent activation constant of 60 + 4 # M was determined from double reciprocal plots (Fig. 4, insert). In the presence of 100 /.~M NADH, the stimulating effect of CoA was 16-17-fold and the apparent activation constant decreased to 29 + 3 /~M. The extent of increase in the esterase reaction rate approached 32-fold in the presence of both effectors (basal activity of 0.4 nmol/min in the absence of CoA and NAD +, activity of 12.8 nmol/min in the presence of CoA and NAD+); a rate close to the acetylation reaction measured from the initial burst data. NAD +, in contrast to NADH, was practically without effect on the ability of CoA to deacetylate MMSDH (Fig. 4). Moreover, CoA had some inhibitory effect on the ability of NAD + to stimulate the hydrolysis of p-nitrophenyl acetate.
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TABLE I Synthesis of acetyI-CoA from p-nitrophenyl acetate a Additions
p-Nitrophenol released (nmol)
AcetyI-CoA synthesized (nmol)
CoA CoA + NADH
91 _+4 86 +_3
95 +_ 8 75 _+10
MALONATE
SEMIALDEHYOE
/
" The products of the esterase reaction were determined in a ~eaction mixture (1 ml) (containing 0.3-0.5 mg MMSDH, 250 nmol p-nitrophenyi acetate, 100 nmol CoA and, where indicated, 100 nmol NADH as described in Materials and Methods. Values are the mean _-!:.S.E. for three determinations.
tZ !
5
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5
10
TIME, MIN. Fig. 5. Inhibition of esterase activity by malonate semialdehyde. Assay conditions were essentially as in Fig. 4. NAD ÷, (100 /zM), CoA (100/~M) and malonate semialdehyde (1 mM) were added as indicated by the arrows.
Malonate semialdehyde inhibits the esterase reaction and p-nitrophenyl acetate inhibits the dehydrogenase reaction Malonate semialdehyde (1 mM), a substrate for the dehydrogenase activity of the enzyme, effectively inhibited ester hydrolysis in the presence of NAD ÷ and CoA. Fig. 5 shows the pattern of malonate semialdehyde inhibition at a concentration of NAD + close to its activation constant (100 izM) and a CoA concentration of 100/.tM. p-Nitrophenyl acetate efficiently inhibited the dehydrogenase reaction catalyzed by MMSDH. The inhibitory constant for 50% inhibition (IC50) was 150 +_ 7 100
Determhzation of the product of the esterase reaction proceeding in the presence of CoA Acetyl-CoA was formed as a product when MMSDH is incubated with p-nitrophenyl acetate and CoA (Table I). The amount of acetyl-CoA synthesized was nearly equal to the amount of p-nitrophenol released. These data indicate that the acyl intermediate formed from p-nitrophenyl acetate is subject of transacetylation in the presence of CoA. NADH, shown above to be an activator of the esterase reaction, did not interfere with the synthesis of acetyl-CoA (Table I), Discussion
!
50
I 500
/~M measured with 50 tiM malonate semialdehyde as substrate (Fig. 6). In separate experiments, pnitrophenyl acetate was found to be a competetive inhibitor of the dehydrogenase reaction with K i = 17 _+ 3 /zM (data not shown). The competitive nature of inhibition by p-nitrophenyl acetate together with inhibition of the esterase reaction by malonate semialdehyde provide evidence that p-nitrophenyl acetate is hydrolyzed at the semialdehyde binding site.
1000
p-NWROPHENYL ACETATE, FM Fig. 6. Inhibition of dehydrogenase activity of MMSDH by pnitrophenyl acetate. MMSDH activity was measured in the presence of 50/~M malonate semialdehyde, 300/~M CoA and 0.5 mM NAD + Specific activity of MMSDH was 7.8 U / r a g protein and 0.5 # g of enzyme protein was used in each assay (1 ml). Each point represents mean + S.D. of four determinations.
The ability of rat liver MMSDH to catalyze ester hydrolysis as well as semialdehyde oxidation has been documented in the present paper. The findings that hydrolysis of p-nitrophenyl acetate by MMSIDH was inhibited by malonate semialdehyde, a substrate of dehydrogenase reaction, and conversely that the dehydrogenase reaction was inhibited by p-nitrophenyl acetate provide evidence for ester hydrolysis taking place at the catalytic center for dehydrogenase activity. This conclusion is also in a good agreement with the finding of regulation of esterolytic activity by pyridine nucleotides (Figs. 2 and 3) and CoA (Fig. 4). The activation constant values for NAD + (110 ~M), NADH (10 /zM) and CoA (29 tzM), determined for the esterase reaction, were reasonably close t o K m values for NAD ÷ (150 t~M) and CoA (30 #M) and the K i value for NADH (3.1 /zM) determined for the dehydrogenase reaction [3].
73 Some aspects of the esterase reaction catalyzed by MMSDH are similar to previous observations made for ALDH [6] and GAPDH [5]. The stimulatory effects of pyridine nucleotides on esterolytic activity of MMSDH were similar to their effects of ALDH, but the magnitude of these effects was different. In the case of ALDH both NAD + and NADH had exactly the same 2-fold stimulatory effect [6], whereas in the case of MMSDH, NADH was 8-times more potent than NAD +. In greater contrast, NAD + is a strong inhibitor of the esterolytic activity of GAPDH [5]. On the other hand, MMSDH and GAPDH exhibited very similar time courses for p-nitrophenyl acetate hydrolysis. With both enzymes, the reaction occurs with an initial burst of p-nitrophenol production, suggesting hydrolysis of an S-acyl enzyme as the rate-limiting step. In the case of MMSDH, CoA - the natural acceptor of acyl groups accelerated the esterase reaction by 7-fold. In the presence of NADH, the overall enhancement was about 32-fold, i.e. close to the acetylation rate measured from the initial burst of p-nitrophenol formation. However, it was not possible to force the esterase reaction to proceed at a rate faster than the dehydrogenase/CoA ester synthetic reaction. The fact that the stimulated esterase reaction was still slower suggests that the rate limiting step involves the acylation by p-nitrophenyl acetate rather than deacetylation of the enzyme-thioester. If deacetylation were rate limiting, the Vm~, for p-nitrophenyl ester hydrolysis would have been equal to or greater than that found for the dehydrogenase/CoA ester synthetic reaction. Thus, in the absence of CoA and NADH the breakdown of S-acetyl enzyme may be a rate limiting step, whereas in the presence of both effectors the rate limiting step occurs prior to the deacetylation step. The f'mding that rat liver MMSDH catalyzes the hydrolysis of p-nitrophenyl acetate strongly supports the hypothesis that an enzyme-thioester: O
II
EwS--C--R
is an important intermediate in the reaction pathway. Whether the formation or breakdown of an S-acyl enzyme intermediate is the rate limiting step for the dehydrogenase reaction catalyzed by rat liver MMSDH has not been determined. Observations reported here for the esterase activity make it clear that the deacylation reaction is rate limiting in the absence of CoA. As was shown for the analogous enzyme from Pseudomonas aeruginosa [1], the dehydrogenase reaction can proceed in the absence of CoA, but extremely slowly. It is, however, impossible to determine the
rate-limiting step for the overall dehydrogenase reaction proceeding in the presence of CoA because the rate of the deacylation reaction in the presence of NADH and CoA can not be readily estimated. A pre-steady state kinetic investigation of both reactions will be required to unequivocally determine which steps are actually rate limiting. For MMSDH from rat liver [3] and for the analogous enzyme from Pseudomonas aeruginosa [1,2], it is known that the oxidative decarboxylation of malonate and methylmalonate semialdehydes depends on the presence of CoA. Hydrolysis of the acyl enzyme in the presence of NADH and NAD + suggests that acetate or propionate could be products of the reaction. However, this probably does not occur in vivo as long as sufficient CoA is available since the rate of hydrolysis is negligible relative to the total rate of the dehydrogenase reaction. The question of whether hydrolysis could play a significant role in valine disposal under conditions of limited CoA availability is of interest but will require additional investigation.
Acknowledgments We thank Paul Rougraff for providing 3-hydroxyisobutyrate dehydrogenase which was needed for the estimation of MMSDH activity during purification. This work was supported by grants from the U.S. Public Health Services (NIH DK 40441), Showalter Foundation and the Diabetes Research and Training Center of Indiana University School of Medicine (DK 20542). KMP and NYK are postdoctoral fellows of the Indiana Affiliate of the American Heart Association.
References 1 Sokatch, J.R., Sanders, L.E. and Marshall, V.P. (1968) J. Biol. Chem. 243, 2500-2506. 2 Bannerjee, D., Sanders, L.E. and Sokatch, J.R. (1970) J. Biol. Chem. 245, 1828-1835. 3 Goodwin, G.W., Rougraff, P.M., Davis, E.J. and Harris, R.A. (1989) J. Biol. Chem. 264, 14965-14971. 4 Jakoby, W.B. (1963) in The Enzymes (Boyer, P.B., ed.), Vol. 7, pp. 203-214, Academic Press, New York. 5 Feldman, R.I. and Weiner, H. (1972) J. Biol. Chem. 247, 267-272. 6 Harris, J.I., Meriwether, B.P. and Park, J.H. (1963) Nature 198, 154-157. 7 Park, J.H., Meriwether, B.P., Ciodfeld, P. and Cunningham, L.W. (1961) J. Biol. Chem. 236, 136-141. 8 Sidhu, R.S. and Blair, A.H. (1975) J. Biol. Chem. 250, 7894-7898. 9 Decker, K. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. 4, pp. 1988-1993, Academic Press, New York. 10 Hartley, B.S. and Kilby, B.A. (1054) Biochem. J. 56, 288-295. 1| Racker, E. (1955) Physiol. Rev. 35, 1-56.
