Preparation of a NAD(H)-polymer matrix showing coenzyme function of the bound pyridine nucleotide

To a Sepharose gel the pyridine nucleotide NAD(H) has been bound using dicyclohexyl carbodiimide. In order to improve the steric availability of the nucleotide for added soluble enzymes such as dehydrogenases, a spacer molecule, ε-amino caproic acid, was inserted between the carbohydrate matrix and the nucleotide. The obtained preparation contained 56 μmoles NAD⁺/g dry polymer. The obtained matrix-bound NAD(H) was accepted as coenzyme by added lactate dehydrogenase. These preparations were still active after storage for several weeks at 4° C and could be used repeatedly without loss of activity. This represents the first necessary step taken in the preparation of compact closed systems consisting of “enzyme–coenzyme–coenzyme-regenerating enzyme” bound to individual polymer beads; such systems eliminate the need for continuous coenzyme addition.     

Solution conformation of lactate dehydrogenase as studied by saturation transfer ESR spectroscopy.

Several binary and ternary inhibitor and 'dead end' complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) were studied by saturation transfer ESR spectroscopy by means of an active NAD analog, spin-labeled at N6. The mobility of the spin-label depends on the nature of small molecules bound at the remote catalytic end of the coenzyme. The spin-label was found to serve as a reporter group monitoring the conformation of the peptide loop that is folded down over the active cleft in crystals of ternary complexes. The data suggest a fluctuation of the loop between open and closed forms in solution. The structure of the inhibitor molecules has been correlated with their ability to stabilize a more closed conformation of the loop.     

Separation of rat lactate dehydrogenase isoenzyme C4 from other isoenzymes by affinity and ion-exchange chromatography.

Lactate dehydrogenase C, an isoenzyme composed of C polypeptide subunits and found only in mature testes and spermatozoa, differs kinetically, chemically and immunologically from the five common isoenzymes of lactate dehydrogenase, each of which is a tetramer of A and/or B subunits. In the rat lactate dehydrogenase C exists in two molecular forms, isoenzymes C4 and A1C3. In addition to these two forms of lactate dehydrogenase C, rat testicular homogenate contains all the five isoenzymes of A and B type. Purification of isoenzyme C4 requires its separation from the other six isoenzymes, of which isoenzymes A1C3 and A3B1 are the most difficult ones to separate. In the present study isoenzyme A3B1, along with other enzymes, was separated from isoenzyme C4 by AMP-Sepharose chromatography by using a gradient of increasing concentration of NAD+-pyruvate adduct. In the next step, isoenzyme A1C3 was separated from isoenzyme C4 by DEAD-cellulose chromatography, resulting in a pure lactate dehydrogenase isoenzyme C4 preparation.     

Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver

1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.     

Refined crystal structure of dogfish M4 apo-lactate dehydrogenase

The crystal structure of M4 apo-lactate dehydrogenase from the spiny dogfish (Squalus acanthius) was initially refined by a constrained-restrained, and subsequently restrained, least-squares technique. The final structure contained 286 water molecules and two sulfate ions per subunit and gave an R-factor of 0.202 for difraction data between 8.0 and 2.0 A resolution. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.25 A. The elements of secondary structure of the refined protein have not changed from those described previously, except for the appearance of a one-and-a-half turn 3(10) helix immediately after beta J. There is also a short segment of 3(10) helix between beta C and beta D in the part of the chain that connects the two beta alpha beta alpha beta units of the six-stranded parallel sheet (residues Tyr83 to Ala87). Examination of the interactions among the different elements of secondary structure by means of a surface accessibility algorithm supports the four structural clusters in the subunit. The first of the two sulfate ions is in the active site and occupies a cavity near the essential His195. Its nearest protein ligands are Arg171, Asp168 and Asn140. The second sulfate ion is located near the P-axis subunit interface. It is liganded by His188 and Arg173. These two residues are conserved in bacterial lactate dehydrogenase and form part of the fructose 1,6-bisphosphate effector binding site. Two other data sets in which one (collected at pH 7.8) or both (collected at pH 6.0) sulfate ions were replaced by citrate ions were also analyzed. Five cycles of refinement with respect to the pH 6.0 data (25 to 2.8 A resolution) resulted in an R value of 0.191. Only water molecules occupy the subunit boundary anion binding site at pH 7.8. The amino acid sequence was found to be in poor agreement with (2Fobs-Fcalc) electron density maps for the peptide between residues 207 and 211. The original sequence WNALKE was replaced by NVASIK. The essential His195 is hydrogen bonded to Asp168 on one side and Asn140 on the other. The latter residue is part of a turn that contains the only cis peptide bond of the structure at Pro141. The "flexible loop" (residues 97 to 123), which folds down over the active center in ternary complexes of the enzyme with substrate and coenzyme, has a well-defined structure. Analysis of the environment of Tyr237 suggests how its chemical modification inhibits the enzyme.     

Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.     

Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase

The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 ? resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.     

Alteration of coenzyme specificity of lactate dehydrogenase from Thermus thermophilus by introducing the loop region of NADP(H)-dependent malate dehydrogenase

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k cat/Km(coenzyme) was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.     

Mechanistic studies on the rat kidney flavoenzyme L-alpha-hydroxy acid oxidase.

The falvoenzyme L-alpha-hydroxy acid oxidase from rat kidney [T.H Cromartie and C.T. Walsh (1975), Biochemistry 14, 2588] fails to catalyze the elimination of HCl form D,L-beta-chlorolactate, although this compound is a substrate for oxidation by the enzyme. Deuterium isotope effects demonstrate that proton removal from the alpha carbon of alpha-hydroxy acids is fully rate limiting, a finding in agreement with observations on L-lactate dehydrogenase from yeast [F. Lederer (1974), Eur. J. Biochem. 46, 393] which also does not promote elimination from D,L-beta-chlorolactate. Both D-alpha-hydroxy acid oxidase were found to be rapidly and irreversibly inactivated by the acetylenic substrate 1-hydroxy-3-butynoate. The partially purified dehydrogenase was observed to be inactivated within 10 min by 6.8 times 10(-8) M hydroxybutynoate. For the more extensively studied oxidase, inactivation was found to occur after 25 catalytic events, inactivation occurring by covalent addition of the inactivator to the coenzyme. A stoichimometry of one molecule of hydroxybutynoate per flavine was found, and the time course of inactivation was unaffected by the presence of thiols. The oxidase could also be inactivated by prolonged incubation of the enzyme with 2-hydroxy-3-butenoate, and inactivation which could be completely prevented by the presence of thiolds. Since the inactivation with hydroxybutenoate also left the flavine coenzyme unaltered, the inactivation was attributed to Michael addition of nucleophiles on the enzyme of the ketobutenoate product. Several 4-alkyl-substitued 2-hydroxy-3-butynoates were also observed to inactivate the oxidase by both coenzyme modification and random addition to the apoenzyme. It is proposed that the inactivation may occur by nucleophilic addition of a C4 allenic carbanion to the oxidized flavine coenzyme.     

Crystal structure of formaldehyde dehydrogenase from Pseudomonas putida: the structural origin of the tightly bound cofactor in nicotinoprotein dehydrogenases

Formaldehyde dehydrogenase from Pseudomonas putida (PFDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase family. The pyridine nucleotide NAD(H) in PFDH, which is distinct from the coenzyme (as cosubstrate) in typical alcohol dehydrogenases (ADHs), is tightly but not covalently bound to the protein and acts as a cofactor. PFDH can catalyze aldehyde dismutations without an external addition of NAD(H). The structural basis of the tightly bound cofactor of PFDH is unknown. The crystal structure of PFDH has been solved by the multiwavelength anomalous diffraction method using intrinsic zinc ions and has been refined at a 1.65 A resolution. The 170-kDa homotetrameric PFDH molecule shows 222 point group symmetry. Although the secondary structure arrangement and the binding mode of catalytic and structural zinc ions in PFDH are similar to those of typical ADHs, a number of loop structures that differ between PFDH and ADHs in their lengths and conformations are observed. A comparison of the present structure of PFDH with that of horse liver ADH, a typical example of an ADH, reveals that a long insertion loop of PFDH shields the adenine part of the bound NAD(+) molecule from the solvent, and a tight hydrogen bond network exists between the insertion loop and the adenine part of the cofactor, which is unique to PFDH. This insertion loop is conserved completely among the aldehyde-dismutating formaldehyde dehydrogenases, whereas it is replaced by a short turn among typical ADHs. Thus, the insertion loop specifically found among the aldehyde-dismutating formaldehyde dehydrogenases is responsible for the tight cofactor binding of these enzymes and explains why PFDH can effectively catalyze alternate oxidation and reduction of aldehydes without the release of cofactor molecule from the enzyme.     

Kinetic characterization of the [3'-32P]coenzyme A/acetyl coenzyme A exchange catalyzed by a three-subunit form of the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum

The ability of acetyl coenzyme A synthesizing carbon monoxide dehydrogenase isolated from Clostridium thermoaceticum to catalyze the exchange of [3'-32P]coenzyme A with acetyl coenzyme A is studied. This exchange is found to have a rate exceeding that of the acetyl coenzyme A carbonyl exchange also catalyzed by CO dehydrogenase ([1-14C]acetyl coenzyme A + CO in equilibrium acetyl coenzyme A + 14CO). These two exchanges are diagnostic of the ability of CO dehydrogenase to synthesize acetyl coenzyme A from a methyl group, coenzyme A, and carbon monoxide. The kinetic parameters for the coenzyme A exchange have been determined: Km(acetyl coenzyme A) = 1500 microM, Km(coenzyme A) = 50 microM, and Vmax = 2.5 mumol min-1 mg-1. Propionyl coenzyme A is shown to be a substrate (Km approximately 5 mM) for the coenzyme A exchange, with a rate 1/15 that of acetyl coenzyme A, but is not a substrate for the carbonyl exchange. CO dehydrogenase capable of catalyzing both these two exchanges, and the oxidation of CO to CO2, is isolated as a complex of molecular weight 410,000 consisting of three proteins in an alpha 2 beta 2 gamma 2 stoichiometry. The proposed gamma subunit, not previously reported as part of CO dehydrogenase, copurifies with the enzyme and has the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the disulfide reductase previously separated from CO dehydrogenase in a final chromatographic step.     

The structural basis of substrate activation in yeast pyruvate decarboxylase. A crystallographic and kinetic study.

The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Structural differences between apo- and holoenzyme of horse liver alcohol dehydrogenase.

The three-dimensional structure of a ternary complex of horse liver alcohol dehydrogenase with reduced nicotinamide adenine dinucleotide and the inhibitor dimethyl sulfoxide has been determined to 4.5 A resolution independently of the apoenzyme structure. The electron density maps of both structures have been compared. The two coenzyme binding domains which form the center of the dimer molecular have retained their conformation and orientation within the molecule whereas the catalytic domains rotate and narrow the cleft between the domains. The active site becomes shielded from the solution by a combination of this rotation, local movements of a loop from residues 53 to 57 and coenzyme and substrate binding. Both subunits bind coenzyme and inhibitor to the same extent. The nicotinamide ring of the coenzyme is positioned close to the active zinc atom and the inhibitor is bound to this zinc atom. The difference between the two crystallographically independent subunits is small. The proposed mechanisms of action for the enzyme based on the apoenzyme structure are confirmed by the present investigation.     

Acetaldehyde coenzyme A dehydrogenase of Escherichia coli.

Mutants of Escherichia coli (adh) in which alcohol dehydrogenase is derepressed under aerobic conditions were also found to overproduce acetaldehyde coenzyme a dehydrogenase. However, acetaldehyde coenzyme A dehydrogenase was induced by ethanol or acetaldehyde and subject to strong catabolite repression, whereas alcohol dehydrogenase was little affected by these conditions. Mutants no longer able to use ethanol as carbon source were isolated from an adh strain. Some of these mutants were revertants at the adh locus and no longer produced either alcohol dehydrogenase or acetaldehyde coenzyme A dehydrogenase. Others, designated acd, were found to lack only acetaldehyde coenzyme A dehydrogenase. The acd mutation was located at min 62 of the E. coli genetic map, the gene order being thyA-lysA-acd-serA-fda. Isolation of Tn10 insertions cotransducible with acd greatly simplified the mapping procedure.     

The preparation of crystalline human l-lactate–nicotinamide–adenine dinucleotide oxidoreductase isoenzyme 1 involving preparative polyacrylamide-gel electrophoresis

A method is presented for the preparation of human heart lactate dehydrogenase (l-lactate-NAD(+) oxidoreductase; EC 1.1.1.27) isoenzyme 1; this involves the use of polyacrylamide-gel electrophoresis as a preparative step. The yield was about 10% with a final specific activity of 220 units/mg of protein, one unit being defined as the amount of enzyme catalysing the oxidation of 1mumol of NADH/min at 25 degrees C, in the presence of 0.33mm-pyruvate. The crystalline preparation contained less than 2% of the other isoenzymes, was homogeneous in the ultracentrifuge and showed only a trace of protein contamination on polyacrylamide-gel electrophoresis. Some properties of the crystalline isoenzyme are reported; E(1%) (1cm)=13.2 at 280nm, s(0) (20,w)=7.43S, pI=4.6, and the apparent K(m) for pyruvate=1.02x10(-4)m. The human isoenzyme and the isoenzyme from pig heart differ with respect to amino acid composition, electrophoretic mobility and solubility. It is possible that these differences do not involve the active site, or sites, but are due to changes in amino acid residues elsewhere in the molecule. The importance of purified human LDH-1 isoenzyme with regard to enzyme radioimmunoassay is emphasized.     

Crystal Structures and Molecular Dynamics Simulations of Thermophilic Malate Dehydrogenase Reveal Critical Loop Motion for Co-Substrate Binding

Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60°C and specific activity of about 2,600 U/mg with optimal temperature of 90°C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme''s activity.     

EDTA-binding and acylation of the Ca2+-sensitive photoprotein aequorin

The rate of phosphorylation and concomitant inactivation of purified pig heart muscle pyruvate dehydrogenase complex by intrinsic kinase (EC 2.7.1.99) is markedly accelerated by the addition of coenzyme A to the incubation medium, showing a half-maximum effect at 1.8 μM. The pantetheine moiety is the effective part of the coenzyme A molecule. The free thiol group is prerequisite for the stimulatory action, acetyl-CoA, benzoyl-CoA or CoAS-SCoA being ineffectual. The thiol's specificity is evidenced by showing that dithiothreitol, 2-mercaptoethanol or glutathione up to 5 mM failed to replace coenzyme A. The possibility is considered that coenzyme A might act as a physiological modifier of pyruvate dehydrogenase kinase activity.     

Chemical modification of aldehyde dehydrogenase by a vinyl ketone analogue of an insect pheromone.

A major component of the sex pheromone from the tobacco budworm moth Heliothis virescens is a C16 straight-chain aldehyde with a single unsaturation at the eleventh position. The sex pheromones are inactivated when metabolized to their corresponding acids by insect aldehyde dehydrogenase. During this investigation it was demonstrated that the C16 aldehyde is a good substrate for human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 with Km and Kcat. values at pH 7.0 of 2 microM and 0.4 mumol of NADH/min per mg and of 0.6 microM and 0.24 mumol of NADH/min per mg respectively. A vinyl ketone analogue of the pheromone inhibited insect pheromone metabolism; it also inactivated human aldehyde dehydrogenase. Total inactivation of both isoenzymes was achieved at stoichiometric (equal or less than the subunit number) concentrations of vinyl ketone, incorporating 2.1-2.6 molecules/molecule of enzyme. Substrate protection was observed in the presence of the parent aldehyde and 5'-AMP. Peptide maps of tryptic digests of the E2 isoenzyme modified with 3H-labelled vinyl ketone showed that incorporation occurred into a single peptide peak. The labelled peptide of E2 isoenzyme was further purified on h.p.l.c. and sequenced. The label was incorporated into cysteine-302 in the primary structure of E2 isoenzyme, thus indicating that cysteine-302 is located in the aldehyde substrate area of the active site of aldehyde dehydrogenase. Affinity labelling of aldehyde dehydrogenase with vinyl ketones may prove to be of general utility in biochemical studies of these enzymes.