Identification of the cysteine residue in the active site of horse liver mitochondrial aldehyde dehydrogenase

Aldehyde dehydrogenase catalyzes the oxidation of aldehydes to acids through the formation of a covalent intermediate. It has been postulated that a cysteine residue could be acting as the active site nucleophilic group. Although N-ethylmaleimide was found to react with many cysteines it was possible by doing the reaction in the presence of chloral hydrate, a substrate analog which functions as a competitive inhibitor, to label cysteine at position 49 in the horse liver mitochondrial enzyme. The dehydrogenase activity was lost as the residue was modified, consistent with the possibility that the residue was an integral component of the active site of the enzyme. Cysteines at positions 162 and 369 also could be modified. It is suggested that cysteine 162 may function as part of a site capable of hydrolyzing nitrophenyl acetate. Details of the second site will appear in the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1218-1222). It appeared that the substrate-binding domain was in the N-terminal portion of the enzyme while the coenzyme binding domain was in the C-terminal portion. During this investigation 133 of the 500 residues of the horse liver enzyme were sequenced. These showed about 95% sequence identity with those of the human enzyme. Inasmuch as both beef and rat liver enzymes also share 95% identity with the human enzyme it can be expected that the results found with the horse liver enzyme can be applicable to all mammalian aldehyde dehydrogenase.     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

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 acyl 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 acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.     

High concentrations of aldehydes slow the reaction of cytoplasmic aldehyde dehydrogenase with thiol-group modifiers.

High concentrations of aldehydes slow the inactivation of cytoplasmic aldehyde dehydrogenase by disulfiram and also slow the reaction of the enzyme with 2,2'-dithiodipyridine. It is concluded that a low-affinity aldehyde-binding site is probably the site at which thiol-group modifiers react with aldehyde dehydrogenase, as well as being the active site for hydrolysis of 4-nitrophenyl acetate.     

Aldehyde dehydrogenase. An enzyme with two distinct catalytic activities at a single type of active site.

The evidence for and against the esterase and dehydrogenase active sites of aldehyde dehydrogenase being topologically distinct is examined. It is found that all the evidence (including all that previously amassed by others in favour of distinct binding domains) is actually consistent with, and in favour of, a single type of catalytic site having both activities. The existence of separate high-Km modulating sites for the enzyme is also questioned.     

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.     

Differences in nucleotide specificity and catalytic mechanism between Vibrio harveyi aldehyde dehydrogenase and other members of the aldehyde dehydrogenase superfamily

The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP(+). The binding of NADP(+) appears to arise from the interaction of the 2'-phosphate of the adenosine moiety of NADP(+) with a threonine (T175) in the nucleotide recognition site just after the beta(B) strand as well as with an arginine (R210) that pi stacks over the adenosine moiety. The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism. However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile. As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile. The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.     

Reversal of part of the aldehyde dehydrogenase reaction pathway during the hydrolysis of an ester.

An aldehyde dehydrogenase from rabbit liver, a homogeneous protein on three distinct polyacrylamide-gel systems, has an associated 4-nitrophenyl esterase activity. At pH 7.0 in the presence of 80 micrometer-NADH and 800 micrometer-4-nitrophenyl acetate the enzyme produces NAD+ and a stoicheiometric amount of an aldehyde, as well as hydrolysing the ester. On this and other evidence it is proposed that ester hydrolysis occurs at the usual active site of the enzyme.     

A histidine residue in the catalytic mechanism distinguishes Vibrio harveyi aldehyde dehydrogenase from other members of the aldehyde dehydrogenase superfamily

Aldehyde dehydrogenases (ALDHs) catalyze the transfer to NAD(P) of a hydride ion from a thiohemiacetal derivative of the aldehyde coupled with a cysteine residue in the active site. In Vibrio harveyi aldehyde dehydrogenase (Vh-ALDH), a histidine residue (H450) is in proximity (3.8 A) to the cysteine nucleophile (C289) and is thus capable of increasing its reactivity in sharp contrast to other ALDHs in which more distantly located glutamic acid residues are proposed to act as the general base. Mutation of H450 in Vh-ALDH to Gln and Asn resulted in loss of dehydrogenase, (thio)esterase, and acyl-CoA reductase activities; the residual activity of H450Q was higher than that of the H450N mutant in agreement with the capability of Gln but not Asn to partially replace the epsilon-imino group of H450. Coupled with a change in the rate-limiting step, these results indicate that H450 increases the reactivity of C289. Moreover, for the first time, the acylated enzyme intermediate could be directly monitored after reaction with [(3)H]tetradecanoyl-CoA showing that the H450Q mutant was acylated more rapidly than the H450N mutant. Inactivation of the wild-type enzyme with N-ethylmaleimide was much more rapid than the H450Q mutant which in turn was faster than the H450N mutant, demonstrating directly that the nucleophilicity of C289 was affected by H450. As the glutamic acid residue implicated as the general base in promoting cysteine nucleophilicity in other ALDHs is conserved in Vh-ALDH, elucidation of why a histidine residue has evolved to assist in this function in Vh-ALDH will be important to understand the mechanism of ALDHs in general, as well as help delineate the specific roles of the active site glutamic acid residues.     

Differences in nucleotide specificity and catalytic mechanism between Vibrio harveyi aldehyde dehydrogenase and other members of the aldehyde dehydrogenase superfamily

The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP⁺. The binding of NADP⁺ appears to arise from the interaction of the 2′-phosphate of the adenosine moiety of NADP⁺ with a threonine (T175) in the nucleotide recognition site just after the β_B strand as well as with an arginine (R210) that pi stacks over the adenosine moiety. The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism. However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile. As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile. The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.     

Multifunctionality of liver alcohol dehydrogenase: Kinetic and mechanistic studies of esterase reaction

Alcohol dehydrogenase from horse liver is shown to catalyze ester hydrolysis. Nicotinamide coenzymes do not affect the rate of esterolysis. A kinetic approach to study esterase reaction at low substrate to enzyme ratio is described. Kinetic effects of ester structure, temperature, pH, solvent polarity, and ionic strength were investigated. The liver enzyme enhances the rate of esterolysis by lowering activation energy of reaction according to the Uni-Bi kinetic sequence. Two ionizable groups, cysteine and lysine, are tentatively assigned at the esterolytic site of liver alcohol dehydrogenase from pH-rate profiles and chemical modification studies. A plausible mechanism for the esterase reaction proceeds via the acid-assisted nucleophilic catalysis involving the ammonium ion of lysine and the thiolate of cysteine in the acyl-oxygen cleavage.     

Chemical studies of high-Km aldehyde dehydrogenase from rat liver mitochondria

Studies of pH-dependent kinetics implicate two ionizable groups in the dehydrogenase and esterase reactions catalysed by high-Km aldehyde dehydrogenase from rat liver mitochondria. Sensitized photooxidation completely arrests the bifunctional activities of the dehydrogenase. Carboxamidomethylation abolishes the dehydrogenase activity, whereas acetimidination eliminates the esterase activity. These results suggest that histidine (pKa near 6) and cysteine (pKa near 10) are likely the catalytic residues for the dehydrogenase activity, while the esterase activity is functionally related to histidine (pKa near 7) and a residue with the pKa value of 10-11. The two residues, a carboxyl group and an arginine, that discriminate between NAD+ and NADP+ are present at the coenzyme binding site of the mitochondrial high-Km aldehyde dehydrogenase from rat liver.     

Bromoacetophenone as an affinity reagent for human liver aldehyde dehydrogenase

Human liver aldehyde dehydrogenase isozymes E1 and E2 (EC 1.2.1.3) are both completely and irreversibly inactivated by bromoacetophenone (2-bromo-1-phenylethanone). Steady-state kinetics with both acetophenone and chloroacetophenone indicated interaction with the same enzyme form as the aldehyde substrate. Saturation kinetics with chloroacetophenone and bromoacetophenone indicated interaction at a specific site on the enzyme surface and gave a dissociation constant similar to that from steady-state kinetics, suggesting that the same processes were being observed by both methods and that the active site may be involved. Protection against inactivation was afforded by chloral and NAD together. Stoichiometry of inactivation showed the first 2 equiv per tetramer to abolish the majority of catalytic activity; 4 equiv inactivated both isozymes with complete loss of esterase, NAD-stimulated esterase, and dehydrogenase activities. Peptide mapping of enzyme modified with [carbonyl-14C]bromoacetophenone of CNBr digests (E1) and tryptic digests (E1 and E2) showed one peptide to be preferentially labeled. The above results together with the similarity of bromoacetophenone to the substrate benzaldehyde suggest bromoacetophenone may react with a residue in the active site of aldehyde dehydrogenase. Amino acid analysis of the labeled E1 tryptic fragment indicated reaction with a different peptide from that with which iodoacetamide reacts.     

Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase.

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.     

Haloenol lactones as inactivators and substrates of aldehyde dehydrogenase

Human aldehyde dehydrogenase (EC 1.2.1.3) isozymes E1 and E2 were irreversibly inactivated by stoichiometric concentrations of the haloenol lactones 3-isopropyl-6(E)-bromomethylene tetrahydro-pyran-2-one and 3-phenyl-6(E)-bromomethylene tetrahydro-pyran-2-one. No inactivation occurred with the corresponding nonhalogenated enol lactones. Both the dehydrogenase and esterase activities were abolished. Activity was not regained on dialysis or treatment with 2-mercaptoethanol. The inactivation was subject to substrate protection: NAD afforded protection which increased in the presence of the aldehyde-substrate competitive inhibitor chloral. Saturation kinetics gave positivey-axis intercepts, allowing the determination of binding constants. Inactivation stiochiometry determined with¹⁴C-labeled 3-(1-naphthyl)-6(E)-iodomethylene tetrahydropyran-2-one was found to correspond to the active-site number. The nonhalogenated lactone, 3-(1-naphthyl)-6(E)-methylene tetrahydropyran-1-one was shown to be a substrate for aldehyde dehydrogenase via its esterase function. Inactivation and enzymatic hydrolysis occurred within a similar time frame. Opening of the lactone ring to form enzyme-acyl intermediate with active site cysteine appears to be a necessary prerequisite to inactivation, since halogen in the lactone ring is nonreactive. Thus, the inactivation of aldehyde dehydrogenase by haloenol lactones is mechanism-based. Inactivation by haloenol lactones occurs in a manner analogous to that of chymotrypsin with which aldehyde dehydrogenase shares esterase activity and binding of haloenol lactones at the active site.     

Modification of aldehyde dehydrogenase with dicyclohexylcarbodiimide: Separation of dehydrogenase from esterase activity

Dehydrogenase activity of the cytoplasmic (E1) isozyme of human liver aldehyde dehydrogenase (EC 1.2.1.3) 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. TheK _m for p-nitrophenyl acetate was increased, but physicochemical properties remained unchanged. The selective inactivation of dehydrogenase activity 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 E1 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.     

Human liver aldehyde dehydrogenase. Esterase activity.

Human liver aldehyde dehydrogenase has been found to be capable of hydrolyzing p-nitrophenyl esters. Esterase and dehydrogenase activities exhibited identical ion exchange and affinity properties, indicating that the same protein catalyzes both reactions. Competitive inhibition of esterase activity by glyceraldehyde and chloral hydrate furnished evidence that p-nitrophenyl acetate was hydrolyzed at the aldehyde binding site for dehydrogenase activity. Pyridine nucleotides modified esterase activity; NAD+ accelerated the rate of p-nitrophenyl acetate hydrolysis more that 5-fold, whereas NADH increased activity by a factor of 2. Activation constants of 117 muM for NAD+ and 3.5 muM for NADH were obtained from double reciprocal plots of initial rates as a function of modifier concentration at pH 7. The kinetics of activation of ester hydrolysis were consistent with random addition of pyridine nucleotide modifier and ester substrate to this enzyme.     

The reaction of N-ethylmaleimide at the active site of succinate dehydrogenase.

Since 1938 mammalian succinate dehydrogenase has been thought to contain thiol groups at the active site. This hypothesis was questioned recently, because irreversible inhibition by bromopyruvate and N-ethylmaleimide appeared not to satisfy the requisite criteria for reaction at the active site. These recent observations of incomplete inactivation of succinate dehydrogenase by N-ethylmaleimide and incomplete protection by substrates can, however, be explained adequately by the presence of oxalacetate and other strong competitors of the inactivation process in the enzyme used in these studies. Substrates, competitive inhibitors, and anions which activate succinate dehydrogenase protect the enzyme from inhibition by N-ethylmaleimide. Inhibition of succinate dehydrogenase by N-ethylmaleimide involves at least two second order reactions which are pH dependent, with pKa values of 8.0 to 8.2. This pH dependence, the known reactivity of N-ethylmaleimide toward thiols, and the protection by substrate and competitive inhibitors indicate that sulfhydryl residues are required for catalytic activity and perform an essential, not secondary, role in the catalysis. Just as the presence of tightly bound oxalacetate prevents inhibition by N-ethylmaleimide, alkylation of the sulfhydryl residue(s) at the active site prevents the binding of [14C]oxalacetate. Thus, these thiol groups at the active site also may be the site of tight binding of oxalacetate during the activation-deactivation cycle.     

The action of cytoplasmic aldehyde dehydrogenase on methyl p-nitrophenyl carbonate and p-nitrophenyl dimethylcarbamate.

Cytoplasmic aldehyde dehydrogenase catalyses the hydrolysis of methyl p-nitrophenyl (PNP) carbonate at an appreciable rate that is markedly stimualted by NAD+ or NADH. The nuleotides accelerate the rate-limiting hydrolysis of the acyl-enzyme intermediate while slowing the observed burst of p-nitrophenoxide production. With PNP dimethylcarbamate the enzyme catalyses the slow release of approx. 1 molecule of p-nitrophenoxide per tetrameric enzyme molecule; during the reaction the enzyme becomes effectively inactivated, as the rate of hydrolysis of the acyl-enzyme is virtually zero. The presence of NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol or disulfiram slows the reaction of enzyme with PNP dimethylcarbamate. The reaction appears to be dependent on a group of pKa 7.6, possibly a cysteine residue. The effect of PNP dimethylcarbamate on the dehydrogenase activity of the enzyme is consistent with there being a single type of active site for the enzyme's dehydrogenase and esterase activities. Steric and electronic factors that govern reaction of the enzyme with PNP substrates are discussed.     

Interaction of human aldehyde dehydrogenase with aromatic substrates and ligands

The substrate benzaldehyde (but not propionaldehyde) could elute aldehyde dehydrogenase from a p-hydroxyacetophenone-affinity column, and inhibit the esterase activity (K(i)=47 microM), indicating that this simple aromatic aldehyde binds to the free enzyme and possibly in the substrate-binding site. Thus, the kinetic mechanism for aldehyde dehydrogenase might be dependent upon which aldehyde is used in the reaction. Chloramphenicol which also elutes the enzyme from the affinity column, shows a discriminatory effect by inhibiting the ALDH1 oxidation of benzaldehyde and activating that of propionaldehyde while showing no effect when assayed with hexanal or cyclohexane-carboxaldehyde. Chloramphenicol is an uncompetitive inhibitor against NAD when benzaldehyde is the substrate. We propose that this drug might interact with both the benzaldehyde and NAD binding sites.