Absence Of Coenzyme Research Articles

Electronic absorption and EPR spectroscopy of copper alcohol dehydrogenase: pink, violet and green forms at a Type 1 copper center analog

Blue and non-blue states of the copper center in copper-substituted alcohol dehydrogenase (EC 1.1.1.1) can be attained by coenzyme binding and / or ligand binding to the copper ion. Copper alcohol dehydrogenase has been studied by electronic absorption, CD and EPR spectroscopy in the presence and absence of coenzyme. On the basis of previous work on blue (Type 1) copper proteins with a CuSS ∗ N 2 chromophore the assignment of charge transfer transitions in copper alcohol dehydrogenase is discussed. The latter contains a CuS 2N(OH 2 unit in the ligand-free protein and a CuS 2N 2 unit in the ternary complex with NAD + and pyrazole. It is proposed that the energy of the charge transfer transitions can be used as a structural marker in combination with EPR data. A comparison is made between the spectroscopic properties of the ternary complex of copper alcohol dehydrogenase and the copper centers in stellacyanin and cytochrome- c oxidase (Cu A) in order to test the validity of recent structural models of the type CuS 2N 2, i.e., a cupric ion coordinated to two thiolate ligands. Finally, a close resemblance between the electronic absorption spectra of copper alcohol dehydrogenase and those of other variants of Type 1 copper centers such as the ‘unusual’ copper center of nitrous oxide reductase is noted as an indication of similar coordination environments.

Hasegawaea gen. nov., an ascosporogenous yeast genus for the organisms whose asexual reproduction is by fission and whose ascospores have smooth surfaces without papillae and which are characterized by the absence of coenzyme Q and by the presence of linoleic acid in cellular fatty acid composition.

Hasegawaea gen. nov., an ascosporogenous yeast genus for the organisms whose asexual reproduction is by fission and whose ascospores have smooth surfaces without papillae and which are characterized by the absence of coenzyme Q and by the presence of linoleic acid in cellular fatty acid composition.

Reduction of cytochrome b in mitochondria from yeast lacking coenzyme Q.

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase but had comparable amounts of cytochromes b and c1 as wild-type mitochondria. Addition of succinate to the mutant mitochondria resulted in a slight reduction of cytochrome b; however, the subsequent addition of antimycin resulted in a biphasic reduction of cytochrome b, leading to reduction of 68% of the total dithionite-reducible cytochrome b. No "red" shift in the absorption maximum was observed, and no cytochrome c1 was reduced. The addition of either myxothiazol or alkylhydroxynaphthoquinone blocked the reduction of cytochrome b observed with succinate and antimycin, suggesting that the reduction of cytochrome b-562 in the mitochondria lacking coenzyme Q may proceed by a pathway involving cytochrome b at center o where these inhibitors block. Cyanide did not prevent the reduction of cytochrome b by succinate and antimycin the the mutant mitochondria. These results suggest that the succinate dehydrogenase complex can transfer electrons directly to cytochrome b in the absence of coenzyme Q in a reaction that is enhanced by antimycin. Reduced dichlorophenolindophenol (DCIP) acted as an effective bypass of the antimycin block in complex III, resulting in oxygen uptake with succinate in antimycin-treated mitochondria. By contrast, reduced DCIP did not restore oxygen uptake in the mutant mitochondria, suggesting that coenzyme Q is necessary for the bypass. The addition of low concentrations of DCIP to both wild-type and mutant mitochondria reduced with succinate in the presence of antimycin resulted in a rapid oxidation of cytochrome b perhaps by the pathway involving center o, which does not require coenzyme Q.

On the nature of the ‘nothing dehydrogenase’ reaction

The biochemical mechanism underlying the 'nothing dehydrogenase' reaction during the histochemical demonstration of dehydrogenases using tetranitro BT as the final electron acceptor has been investigated in unfixed, frozen rat liver sections. The reaction is stronger with NAD+ than either with NADP+ or in the absence of coenzyme. As much as 50% of the reaction is due to lactate dehydrogenase converting endogenous lactate and is largely inhibited by pyruvate. No NAD+-dependent alcohol dehydrogenase activity was detected at pH 7.45, the pH used for the incubations. The coenzyme-independent activity may be caused by SH-groups present in proteins and compounds like glutathione and cysteine and can be inhibited by N-ethylmaleimide and p-chloromercuribenzoic acid. It was also found that the 'nothing dehydrogenase' reaction mainly occurs during the first few minutes of incubation, levelling off quickly to a slow rate. When studying the kinetics of dehydrogenase reactions with tetrazolium salts, it should be realized that the 'nothing dehydrogenase' reaction, which as a whole is nonlinear with time, can interfere seriously with the dehydrogenase reaction to be analysed and may yield initial reaction rates that are too high. The findings of the present study reveal the nature of the reactions used for detection of necrosis in tissues with tetrazolium salts.

Binary and ternary complexes of malate dehydrogenase with substrates and substrate analogs

Hydroxypyrenetrisulfonate binds to pig mitochondrial malate dehydrogenase ( l-malate:NAD + oxidoreductase, EC 1.1.1.37) in the presence and absence of coenzymes with a stoichiometry of one dye molecule/enzyme subunit. Binding is competitive with substrates and known substrate analogs as well as with squaric acid, a newly detected analog forming a fernary complex with enzyme/NAD + similar to enzyme/NAD +/sulfite. Displacement of hydroxypyrenetrisulfonate by substrates and analogs was used to determine dissociation constants of binary and ternary complexes. Binary complexes form with dissociation constants of about 10 mM. They may be important for kinetic studies at high substrate concentrations where oxaloacetate inhibition and malate activation have been described.

Isoelectric focusing studies of aldehyde dehydrogenases from mouse tissues: Variant phenotypes of liver, stomach and testis isozymes

1.1. Isoelectric focusing techniques (IEF) were used to examine the tissue distribution and genetic variability of aldehyde dehydrogenases (AHDs) from inbred strains of mice.2.2. Twelve zones of AHD activity were resolved which were differentially distributed between tissues. Liver extracts exhibited highest activity for most enzymes, with the exception of isozymes found in stomach (AHD-4) and testis (AHD-4 and AHD-6).3.3. Genetic variants for AHD-1 (liver mitochondrial isozyme) and AHD-4 (stomach isozyme) were examined from inbred strains and F1 hybrid animals. The results were consistent with dimeric subunit structures (designated as A2 and D2 isozymes respectively).4.4. IEF patterns for activity variants of testis-specific AHD-6 were identical, with 3-banded phenotypes being observed.5.5. pI values for the AHD forms as well as for aldehyde oxidase and xanthine oxidase isozymes, which stain in the absence of coenzyme, were reported.

Thermodynamic bases for fatty acid ethyl ester synthase catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters.

Myocardial homogenates rapidly synthesize fatty acyl ethyl esters from nonesterified fatty acid and ethanol in the absence of coenzyme A or ATP, and the enzyme catalyzing this reaction, fatty acid ethyl ester synthase, has been purified 5400-fold to homogeneity [Mogelson, S., & Lange, L. G. (1984) Biochemistry (preceding paper in this issue)]. To define the factors permitting this de novo synthesis of ester bonds and the consequent accumulation of fatty acyl ethyl esters in myocardium, we determined thermodynamic parameters relevant to the kinetics and equilibria of this reaction and specifically characterized (1) the rates of synthesis of ethyl oleate, in both the presence and absence of purified enzyme catalyst, and (2) the physical properties of the product, ethyl oleate, in an aqueous milieu. Compared to the reaction of ethanol and oleate in the absence of catalyst, fatty acid ethyl ester synthase enhanced the rate of ethyl oleate synthesis by reducing the free energy of activation (delta G) from 32.5 to 19.9 kcal/mol, effected in large part by a positive entropy shift, delta Senz - delta S uncat = 23.9 cal/(mol.deg). Rate constants in the presence and absence of enzyme at 37 degrees C were 6 X 10(-2) s-1 and 7.8 X 10(-11) M-1 s-1, respectively, indicating a catalytic power of at least 10(8)M for this enzyme. Kinetic data indicated an enzymatic Vmax of 1.25 nmol/(mg.s) (37 degrees C). The equilibrium constant was calculated for the reaction oleate + ethanol in equilibrium ethyl oleate and was 0.095 M-1 at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial purification and product characterization of fatty acid ethyl ester synthases in rabbit myocardium

Homogenates of rabbit ventricular myocardium synthesize fatty acid ethyl esters using as substrates nonesterified fatty acid and ethanol in the absence of coenzyme A and ATP. This catalytic activity resides in two soluble cytosolic enzymes accounting for 19 and 81% of total fatty acid ethyl ester synthetic capability. These enzymes have been separated and partially purified by anion exchange chromatography. Gas chromatographic/mass spectrometric analyses of the catalytic products formed by these enzymes from nonesterified fatty acid and ethanol confirm their identity as ethyl esters of fatty acids. Kinetic studies indicate apparent K m values for ethanol of 0.65 M and 0.75 M for the minor and major activities, respectively. These data confirm the presence of a myocardial pathway for nonoxidative ethanol metabolism and for a metabolism of fatty acids independent of coenzyme A.

Fluorescence investigations of coenzyme and substrate interactions in mannitol-1-phosphate dehydrogenase ofEscherichia coli

Coenzyme and substrate interactions with mannitol-1-phosphate dehydrogenase fromEscherichia coli (a dimer of MW 45,000) have been studied by fluorescence spectroscopy. NAD+ quenches the fluorescence emission of the protein tryptophan residues; shifting the excitation wavelength from 280 to 290 nm results in an increase in this quenching and a red shift in the emission maximum. NAD+ also quenches the fluorescence of covalently attached pyridoxyl phosphate, and this quenching is accompanied by a spectral broadening above 425 nm. Fructose-6-phosphate increases the binding of NAD+, but causes a slight reduction in the quenching of the tryptophan fluorescence observed at saturating levels of coenzyme, and reverses the NAD+-induced broadening in the pyridoxyl phosphate emission spectrum. NADH quenches the protein emission much less than NAD+; this quenching is not changed by shifting the excitation wavelength and is not affected by the presence of bound mannitol-1-phosphate. Titrations monitoring the quenching by NADH indicate a single class of NADH binding sites, while titrations monitoring NADH fluorescence suggest that coenzyme fluorescence is more enhanced when NADH is bound to less than half of the total enzyme subunits, with the emission per NADH molecule bound decreasing as the number of NADH molecules bound increases. In the absence of coenzyme, neither fructose-6-phosphate nor mannitol-1-phosphate have any effect on the protein tryptophan emission; however, both substrates induce specific changes in the emission spectrum of covalently attached pyridoxyl phosphate. These results suggest that the different coenzymes and substrates cause specific conformational changes in mannitol-1-phosphate dehydrogenase.

Structural studies of horse liver alcohol dehydrogenase: Coenzyme, substrate and inhibitor binding

Alcohol dehydrogenase from horse liver has been thoroughly investigated with crystallographic methods. Four different crystal forms of the enzyme have been solved and refined. They show that the enzyme exists in two predominant forms. The open form is found in the absence of coenzyme and has two long deep clefts cutting the enzyme in three units. In the closed form of the enzyme these clefts are closed around the coenzyme and subtrate/inhibitor. Although there are large conformational changes in the enzyme, they are mainly restricted to relative movements of the separate domains. The internal structure of these domains is virtually identical in the open and closed forms. The coenzyme is the main cause of the conformational change and binds with a large number of interactions to the enzyme. About 4% of the enzyme surface is covered by the bound coenzyme. The nicotinamide ring is not bound to the active site zinc atom, but puts one surface of the ring in contact with the zinc coordinated cystein sulphur atoms. The oxygen atom of the substrate binds directly to the zinc atom with the rest of the substrate close to the nicotinamide of the coenzyme. Large substrates extend into a 15–20 Å long hydrophobic channel which opens up towards the solution. The widely used inhibitor pyrazole binds as a bridge between the zinc atom and the nicotinamide ring. Pyrazoles substituted in the 4-position are generally strong inhibitors. This can be properly related to the organization of the substrate channel of the enzyme.

Differential scanning calorimetry of cytoplasmic aspartate transaminase.

Differential scanning calorimetry has been applied to study factors affecting the thermally induced denaturation of cytoplasmic aspartate aminotransferase, a dimeric pyridoxal enzyme. The consequences of binding of coenzyme and substrate derivatives to both the apo and holo forms of the enzyme were investigated and are interpreted in terms of the stabilization of the native form of the enzyme. The binding of pyridoxal phosphate coenzyme increases the thermal stability of the apoenzyme by approximately 27 kcal mol-1 as judged by the change in free energy differences between the native and denatured states of the protein. The stabilization produced by coenzyme binding to the apoprotein appears to be primarily due to the Schiff's base and phosphoryl moieties of the coenzyme; association of the pyridine ring component is without significant structural consequence. Pyridoxal phosphate binding to the subunits of the dimer occurs in a noncooperative fashion as judged by the appearance of transitions unique to the apo, holo, and intermediate enzyme forms in a calorimetric titration. Holoenzyme stability depends on the chemical nature of the catalytically significant group occupying the C-4' position of the bound coenzyme. The stabilization afforded by binding of the aldehyde form (pyridoxal phosphate) which exists as an internal Schiff's base with Lys 258 is diminished when this bond is chemically reduced or when the aldehyde is replaced by an amine (pyridoxamine phosphate). Apoenzyme is also shown to be stabilized by the presence of substrates in the absence of coenzyme. The differential scanning calorimetry results thus confirm previous findings derived from nuclear magnetic resonance studies on the ability of apoenzyme to bind substrates (Martinez-Carrion, M. Cheng, S., and Relimpio, A. (1973) J. Biol. Chem. 248, 2153-2160). Substrates and their analogues perturb the holoenzyme stability and the order of increasing influence on the pyridoxal form of the holoenzyme is aspartate, erythro-hydroxyaspartate, alpha-ketoglutarate, and alpha-methylaspartate. While all these compounds form stable binary enzyme-substrate complexes (Jenkins, W.T., and D'Ari, L. (1966) J. Biol. Chem. 541, 5667-5674), the complex with alpha-methylaspartate produces anomalous changes in the protein structure which are reflected in the calorimetric parameters. This suggests that caution be exercised in the use of analogues as substrate substitutes in crystallographic work. Differential scanning calorimetry also appears as a sensitive method with which to study the stereochemical dependence of ligand binding on enzyme-induced thermal stabilization. This is illustrated by the use of 4-carbon dicarboxylic acids where only those in the conformation favorable for binding are effective in stabilizing the holoenzyme.

Effects of coenzyme analogues on the binding of p-aminobenzoyl-L-glutamate and 2,4-diaminopyrimidine to Lactobacillus casei dihydrofolate reductase.

The binding of p-aminobenzoyl-L-glutamate and 2,4-diaminopyrimidine to dehydrofolate reductase from Lactobacillus casei MTX/R in the presence of a series of co-enzymes and coenzyme analogues has been measured fluorometrically. These two ligands, which can be regarded as "fragments" of the powerful inhibitor methotrexate, have been shown to bind cooperatively in the absence of coenzyme [Birdsall, B., Burgen, A. S. V., Rodrigues de Miranda, J., & Roberts, G. C. K. (1978) Biochemistry 17, 2102], p-amino-benzoyl-L-glutamate binding 58 times more tightly in the presence of 2,4-diaminopyrimidine than in its absence. In the presence of coenzymes, this cooperativity ranges from 1.8- to 428-fold. The effects of coenzymes on individual binding steps range from an 8-fold decrease in binding constant to a 23-fold increase. The structural specificity of these effects are discussed in terms of a model involving ligand-induced conformational changes and compared with the effects on trimethoprim and methotrexate binding described in the preceding paper [Birdsall, B., Burgen, A. S. V., & Roberts, G. C. K. (1980) Biochemistry (first paper of four in this issue)].

Subunit Composition and Partial Reactions of the 2‐Oxoglutarate Dehydrogenase Complex of Acetobacter xylinum

The 2‐oxoglutarate dehydrogenase complex from Acetobacter xylinum consists of three components, 2‐oxoglutarate dehydrogenase (E1), lipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3). The molecular weight of the complex as studied by sedimentation and diffusion is 2.6 × 106. The E1 component and the E2E3 subcomplex were purified by ultracentrifugation and gel filtration after dissociation of the complex at pH 9.8. The E2E3 subcomplex was further dissociated in 1 M NaCl at neutral pH and the components purified by gel filtration.The active E1 component is a tetramer of Mr 360000. The E3 component is a dimer of Mr 103000. The E2E3 subcomplex showed an S20,w value of 36.6 S indicating an Mr around 1.6 × 106. The Mr of E2 was estimated to be 900000. From these data and the chain weights as derived from sodium dodecylsulphate gel electrophoresis it was concluded that the complex consists of 12 E1, 12 E2 and 12 E3 chains.Incorporation of N‐ethyl[2,3‐14C]maleimide in the presence of NADH indicated the presence of 12 reactive SH groups per complex located on the E2 component. Titration of the lipoyl moiety with substoichiometric amounts of N‐ethylmaleimide in the presence of NADH, as assayed by cleavage with 5,5′‐dithio‐bis(2‐nitrobenzoic acid), indicated that the complex contains 12 lipoyl groups of which only one of the two SH groups is reactive towards N‐ethylmaleimide.In agreement with this number, reaction of the complex with 2‐oxo[2‐14C]glutarate yields incorporation of 12–14 mol succinyl groups/mol complex. With non‐saturating concentrations of 2‐oxo‐glutarate, the presence of the allosteric effector AMP or addition of N‐ethylmaleimide was required for maximum incorporation. AMP caused enhancement of the rate of succinylation with cooperative dependence on the AMP concentration, Hill coefficient h= 1.3 at pH 7.4 and h= 2 at pH 8.2. N‐Ethylmaleimide prevented desuccinylation. The rate of succinylation was at least six times slower than the rate of FAD reduction as measured from the decrease in fluorescence in the presence of coenzyme A. This indicates that either coenzyme A participates in the succinylation of the lipoyl moiety or that the formation of succinyllipoate is a side reaction of the catalytic pathway, which takes only place in the absence of coenzyme A.

Combined coenzyme-substrate analog of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases

Two diastereomeric nicotinamide adenine dinucleotide (NAD+) derivatives were synthesized in which the substrates of (S)-and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes and intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analogue composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two (R)-lactate specific dehydrogenases, however, do not catalyze a similar reaction in either one of the two diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.

Purification and properties of allelic lactate dehydrogenase isozymes at the B 2 locus in rainbow trout, Salmo gairdneri

1. 1. The homotetrameric lactate dehydrogenase (LDH) isozymes, B 4 2′ and B 4 2″, from homozygous rainbow trout livers have been purified to homogeneity by affinity chromatography using a Sepharose-linked oxamate ligand. 2. 2. The B 4 2″ isozyme has been found: (a) to have increased K m (pyr) and K i (lac) values, (b) to bind pyruvate strongly in the absence of coenzymes with a binding constant of 4.02 × 10 4 M −1 under conditions where other LDH isozymes showed no binding, and (c) to give a nonlinear noncompetitive lactate product inhibition pattern compared to a normal linear noncompetitive inhibition for the B 4 2′ isozyme. 3. 3. Different kinetic mechanisms have been proposed for the two isozymes and an adaptive functional significance has been discussed.

Effect of coenzymes and temperature on the process of in vitro refolding and reassociation of lactic dehydrogenase isoenzymes

Dissociation and deactivation of the H4 and M4 isoenzymes of lactic dehydrogenase in strong denaturants may be reversed with a yield of reactivation up to 100%. The products of reconstitution are indistinguishable from the native enzymes as far as the Michaelis constants and the dissociation constants for substrate and coenzyme as well as spectral and hydrodynamic properties are concerned. The presence of NAD+ and NADH does not affect either the conformational state of the product of reconstitution, or the kinetics of reactivation, using the pure apoenzymes as a reference. At 20 degrees C the kinetics of reactivation for LDH-M4 in the presence and absence of coenzyme may be quantitatively described by a second-order rate equation (k2 = 23.4 +/- 2.6 mM-1S-1) while LDH-H4 is characterized by a uni-bimolecular reaction sequence (k1 = 1.45 +/- 0.45 X 10(-3)-S-1, k2 = 5 +/- 1 mM-1S-1), in agreement with earlier observations (Rudolph, R., et al. (1977), Biochemistry 16, 3384-3390). Regarding the influence of temperature on the rate of reactivation no significant anomalies are detectable within the range of 0-25 degrees C. The (apparent) activation energies, taken from the linear Arrhenius plots, are 58 kcal/mol for the association reaction of LDH-M4, and 41 kcal/mol for the transconformation reaction of LDH-H4.

Acetylation stoichiometry of Escherichia coli pyruvate dehydrogenase complex

Summary [1- 14 C]Acetyl-pyruvate dehydrogenase complex can be isolated either by gel filtration or by phenol extraction upon reaction of the complex with [2- 14 C]pyruvate in the absence of coenzyme A and NAD. Acetylation by [2- 14 C]pyruvate is thiamine pyrophosphate dependent, all of the radioactivity is covalently bonded to the dihydrolipoyl transacetylase component, and all of the [1- 14 C]acetyl groups are removed by coenzyme A. The radioactivity incorporated corresponds to 9.6 to 10.1 n·mol of acetyl groups per mg dry weight of complex, about four times the number of FAD molecules. Based on a particle molecular weight of 4.6 to 4.8 × 10 6 , this stoichiometry corresponds to 48 acetyl-group binding sites and 12 molecules of FAD per particle of complex.

Coenzyme‐Dependent Conformational Properties of Rat Liver Ornithine Aminotransferase

Two apoenzymes of crystalline ornithine aminotransferase from rat liver were separated by gel filtration. They could also be separated by ultracentrifugation and sucrose density gradient centrifugation. These enzymes have different molecular weights, specific activities and absorption spectra. Their molecular weights are 177 100 ± 7700 (form I) and 105000 ± 6300 (form II) and their specific activities are 11.7 and 23.3 U/mg respectively. The native ornithine aminotransferase and the reconstituted holo‐form I and holo‐form II have similar Km values for l‐ornithine (0.9 to 1.1 mM), Km values for 2‐oxo glutarate (1.4 to 1.8 mM) and Ki; values for l‐valine (4.0 to 6.5 mM). In the absence of coenzyme both enzymes are inert, but form I has an absorption shoulder at 330 nm, like the pyridoxamine 5′‐phosphate form of enzyme. Apo‐form II associates forming a molecule with the same molecular weight as native enzyme of 177000, on addition of the coenzyme, pyridoxal 5′‐phosphate. The molecular weights of each enzyme protomer of the native ornithine aminotransferase and of the two apo‐forms were all found to be 45000 ± 2000 by dodecylsulfate acrylamide gel disc electrophoresis. The amino acids compositions of form I and form II are very similar, and the total number of SH groups/monomer of the native enzyme, apo‐form I and apo‐form II were all 5.

Absence of coenzyme Q 10 as an intrinsic component of aldehyde oxidase

Many investigators have purified an aldehyde oxidase from mammalian livers, and described reactions of this enzyme with diverse substrates. Coenzyme Q 10 was unambiguously identified and found present in some of these enzyme preparations. It was considered that coenzyme Q 10 might participate in the functionality of this enzyme, but the validity of the intrinsic association of coenzyme Q 10 was questioned. We have similarly purified aldehyde oxidase from rabbit livers. No coenzyme Q 10 could be detected under controlled conditions for detecting the presence of coenzyme Q 10. It is concluded that coenzyme Q 10 may be a contaminant of some aldehyde oxidase preparations, and that it is not intrinsic for the functionality of this enzyme.

Histidine residues and the enzyme activity of pig heart supernatant malate dehydrogenase.

1. Supernatant pig heart malate dehydrogenase is completely inhibited by reaction with diethyl pyrocarbonate at pH6.5, when 0.58+/-0.1 residue of ethoxycarbonylhistidine is formed per NADH-binding site. 2. Oxaloacetate and hydroxymalonate protect the enzyme from inhibition in the absence of coenzyme. 3. Limited ethoxycarbonylation does not alter the binding of NADH to the enzyme but prevents the enzyme-NADH complex from interacting with hydroxymalonate in a ternary complex.