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C Woenckhaus

Publications and source records attributed to C Woenckhaus.

At least 37 records · Page 2Linked to original sources
PubMed1996

Photoaffinity labelling of lactate dehydrogenase from pig heart with a bifunctional NAD(+)-analogue.

P1-N6-(4-azidophenylethyl)adenosine-P2-4-(3-azidopyridinio)b utyl diphosphate was synthesized with an [8-14C]adenine label. This bifunctional photoaffinity labelling reagent inactivates lactate dehydrogenase from pig heart upon irradiation with light of wavelength 300-380 nm. Stoichiometry of binding and enzymatic parameters suggest that the analogue is bound to the coenzyme binding site and that adjacent residues are modified. Four radioactive peptides were isolated by reverse-phase HPLC after tryptic digestion of the labelled protein. Amino-acid sequence analysis identified the peptides and correlation with the three-dimensional structure of dogfish lactate dehydrogenase reveals that the peptides correspond to positions affecting the coenzyme binding site, consistent with proper affinity labelling. Two of the peptides, Ile-77 --> Lys-81 and Asp-82 --> Asn-88, are located close to the adenine binding site. Low recovery of Thr-86 in combination with the detection of additional products in the sequence analysis indicates that this residue is modified by the photoaffinity label. The two other peptides (positions 119-124 and 318-328) are located next to the substrate binding site; their label is lost upon treatment with pyrophosphatase, showing that they are linked to the pyridinio moiety of the coenzyme analogue.

Affinity Labels↗
PubMed1989

Preparation and properties of a holo-lactate dehydrogenase enzyme reactor.

NAD+ was the base material for syntheses of coenzyme analogs with reactive groups bound to N6 of the adenine moiety via spacers that are 3-17 A long. These analogs were used for the modification of dehydrogenases. Aromatic imidoesters and acyl azides are suitable reactive groups, which form covalent amidinium or amide bonds with amino acid residues such as the epsilon-amino groups of lysines. The catalytic function of the modified protein decreased only slightly. Coenzymes that are linked via a spacer to carboxyl and amino groups are fixed to the protein by means of carbodiimides and hydroxysuccinimide. Coenzyme-bound aromatic imidoesters with spacer lengths of more than 12 A were incorporated to the extent of 60% at the active site. Aliphatic imidoesters proved to be inefficient for protein modification because of fast hydrolysis. Fixing of coenzyme analogs containing appended carboxyl or amino groups to enzyme in the presence of carbodiimides resulted in a decrease of enzyme activity. Modified lactate dehydrogenase and L-alanine dehydrogenase formed an enzyme reactor for the production of L-alanine in the absence of free NAD+. Both enzymes were cross-linked by dimethyl suberimidate in the presence or absence of NAD+, bis-NAD+, pyruvate, and oxamate. Site-to-site directed cross-linking yielded a reaction mixture from which four protein fractions were obtained by isoelectric focusing; one of these showed a cycling rate of 600 h-1.

Alanine Dehydrogenase↗
PubMed1988

Mitochondrial aldehyde dehydrogenase from horse liver. Correlations of the same species variants for both the cytosolic and the mitochondrial forms of an enzyme.

The primary structure of the mitochondrial form of horse liver aldehyde dehydrogenase has been determined, utilizing peptide analyses and homology with other enzyme forms. The subunit exhibits N-terminal heterogeneity in size similar to that for the corresponding human mitochondrial protein, the longest form having 500 residues. Catalase was identified as a contaminant of the preparations. All four pairs within a set of aldehyde dehydrogenases can now be compared, including the same two species variants (horse and human) for both the cytosolic and mitochondrial enzyme, revealing characteristic differences although Cys-302 and other segments of presumed functional importance are unchanged. The cytosolic and mitochondrial enzymes are clearly different (172 exchanges in the horse pair; 160 exchanges in the human pair) and the mitochondrial forms are more conserved (28 exchanges of 500 residues) than the cytosolic ones (43 exchanges). Distributions of the residue substitutions also differ between the two enzyme types. These results suggest a comparatively distant separation of the cytosolic and mitochondrial enzymes into forms with separate functional constraints that are more strict on the mitochondrial than the cytosolic enzyme. Unexpectedly, positions with residues unique to one of the four enzymes are about twice as common in both of the horse proteins than in either of the human proteins. This difference may reflect a general pattern for human/non-human proteins, showing that not only functional properties of the protein, but also other factors, such as generation time (longer in man than in horse), are important for enzyme divergence.

Aldehyde Dehydrogenase↗
PubMed1986

Kinetics of native and modified liver alcohol dehydrogenase with coenzyme analogues: isomerization of enzyme-nicotinamide adenine dinucleotide complex.

Coenzyme analogues with the adenosine ribose replaced with n-propyl, n-butyl, and n-pentyl groups; coenzyme analogues with the adenosine replaced with 3-(4-acetylanilino)propyl and 6-(4-acetylanilino)hexyl moieties; and nicotinamide mononucleotide, nicotinamide hypoxanthine dinucleotide, and 3-acetylpyridine adenine dinucleotide were used in steady-state kinetic studies with native and activated, amidinated enzymes. The Michaelis and inhibition constants increased up to 100-fold upon modification of coenzyme or enzyme. Turnover numbers with NAD+ and ethanol increased in some cases up to 10-fold due to increased rates of dissociation of enzyme-reduced coenzyme complexes. Rates of dissociation of oxidized coenzyme appeared to be mostly unaffected, but the values calculated (10-60 s-1) were significantly less than the turnover numbers with acetaldehyde and reduced coenzyme (20-900 s-1, at pH 8, 25 degrees C). Rates of association of coenzyme analogues also decreased up to 100-fold. When Lys-228 in the adenosine binding site was picolinimidylated, turnover numbers increased about 10-fold with NAD(H). Furthermore, the pH dependencies for association and dissociation of NAD+ and turnover number with NAD+ and ethanol showed the fastest rates above a pK value of 8.0. Turnover with NADH and acetaldehyde was fastest below a pK value of 8.1. These results can be explained by a mechanism in which isomerization of the enzyme-NAD+ complex (110 s-1) is partially rate limiting in turnover with NAD+ and ethanol (60 s-1) and is controlled by ionization of the hydrogen-bonded system that includes the water ligated to the catalytic zinc and the imidazole group of His-51.

Alcohol Dehydrogenase↗
PubMed1986

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes.

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.

Alanine Dehydrogenase↗
PubMed1985

Characterization of the coenzyme binding site of liver aldehyde dehydrogenase: differential reactivity of coenzyme analogues.

The mitochondrial isozyme of horse liver aldehyde dehydrogenase was labeled with brominated [5-(3-acetylpyridinio)pentyl]diphosphoadenosine. Specific labeling of a coenzyme binding region was proven by an enzymatic activity of the isozyme with the nonbrominated coenzyme derivative, optical properties of the complex, stoichiometry of incorporation, and protection against inactivation. A cysteine residue was selectively modified by the brominated coenzyme analogue and was identified in a 35-residue tryptic peptide. This cysteine residue corresponds to Cys-302 of the cytoplasmic isozyme and has earlier been implicated in disulfiram binding, confirming a position close to the active site. In contrast, the butyl homologue of the coenzyme analogue labels another residue of the mitochondrial isozyme. Thus, in the same isozyme, two residues are selectively reactive. They are concluded to be close together in the tertiary structure and to be close enough to the coenzyme binding site to be differentially labeled by coenzyme analogues differing only by a single methylene group.

Aldehyde Dehydrogenase↗
PubMed1983

Affinity labelling of the allosteric site of the L-lactate dehydrogenase of Lactobacillus casei.

Kinetic investigations employing the substrate analogues 2-oxoglutarate and phospho(enol)pyruvate indicate that the allosteric L-lactate dehydrogenase (EC 1.1.1.27) of Lactobacillus casei has a non-catalytic pyruvate-binding site to which, in addition to pyruvate, the allosteric effector fructose 1,6-bisphosphate can also be found. A modification using the 14C-labelled substrate analogue 3-bromopyruvate induces a loss of regulation by fructose 1,6-bisphosphate. The histidine residue labelled by 3-bromopyruvate is homologous to histidine-188 which is part of the anion-binding site of the non-allosteric vertebrate L-lactate dehydrogenases. Thus, the allosteric site of the allosteric L-lactate dehydrogenases corresponds to the anion-binding site of the non-allosteric vertebrate enzymes.

Affinity Labels↗
PubMed1981

Sulfonate analogues of adenosine nucleotides as inhibitors of nucleotide-binding enzymes.

2-(Adenin-9-yl)ethanesulfonic acid (1), 3-(adenin-9-yl)propanesulfonic acid (2), 9-(5-deoxy-beta-D-ribofuranosyl)-adenine-5'-sulfonic acid (3), and 9-(3-deoxy-beta-D-arabinofuranosyl)adenine-3'-sulfonic acid (4) were prepared by reaction of the corresponding chlorides by sodium sulfite (1-3) or by reaction of an epoxide with sodium hydrogen sulfite (4). They inhibited a typical nucleotide-binding enzyme, horse liver alcohol dehydrogenase, with inhibition constants in the range of 0.18-4.9 mM at pH 8, 25 degrees C.

Adenine Nucleotides↗
PubMed1981

Characterization of a structure close to the coenzyme-binding site of liver aldehyde dehydrogenase.

NAD analogues with the nicotinamide moiety exchanged for acetylpyridino-pentyl or acetylpyridino-butyl groups function as coenzymes in the enzymatic reaction with liver aldehyde dehydrogenase. The corresponding bromoacetyl derivatives bind to the coenzyme-binding site of the enzyme and inactive the protein by covalent modification of single residues close to the active site. Protection by coenzymes and substrate against the inactivation differs slightly for the two coenzyme analogues, suggesting the presence of more than one reactive residue. This is consistent with the results of differential carboxymethylation of cysteine residues of the basic isozyme in the presence and absence of the inhibitor disulfiram. The amino acid sequence around one reactive cysteine residue close to the active site of the acidic isozyme was determined after labeling with the butyl coenzyme analogue. This structure bears no extensive homology to corresponding known structures of dehydrogenases working on other types of aldehyde substrates.

Aldehyde Dehydrogenase↗
PubMed1981

[Diazonium derivatives of ADP for the identification of essential amino acid side chains in the active site of dehydrogenases].

For affinity labeling of NAD-dependent dehydrogenases, dinucleotide analogs were prepared by connecting nitrobenzene or nitrobenzimidazole systems with adenosine diphosphate. The distance between the two parts of the molecule was varied by insertion of propyl, butyl and pentyl chains or ribose. Reduction of the nitro group with hydrazine/Raney nickel yielded the corresponding amino derivatives which were converted to the diazonium salts by nitrous acid. Due to specific linking of ADP moiety to dehydrogenases, the reactive diazonium group combines with nucleophilic amino acid side chains in the active centre of dehydrogenases, the enzymatic activity of which was protected by NAD and NADH. Fluorescence titration experiments proved a linear correlation between incorporation of nucleotide anhydride, residual activity and remaining NADH capacity of the enzymes. The different modified amino acids showed characteristic absorption bands which allowed the identification of the reacting group as well as the estimation of the stoichiometry of the reaction. The latter could be estimated by titration of the enzyme with the diazonium salt. Only in a few cases was the spectrophotometric identification of the modified amino acid side chain uncertain. This fact required enzymatic degradation of the protein followed by electrophoresis and amino acid analysis.

Amino Acids↗
PubMed1980

Correlations between tertiary structure and energetics of coenzyme binding in pig heart muscle lactate dehydrogenase.

Fluorescence, equilibrium dialysis, and microcalorimetric measurements have been performed on complex formation between pig heart muscle lactate dehydrogenase (EC 1.1.1.27) and a series of systematically modified nicotinamide adenine dinucleotide analogues to provide quantitative data for a discussion on energy-structure-function correlations. As a result of these studies, one can draw the conclusion that estimates of the relative stability of enzyme-ligand complexes on the mere basis of structural information on the macromolecule and its complexes with the ligand are likely to neglect contributions to the energy and entropy parameters, which stem from such processes as changes in solvation and conformation of both the free ligand and the macromolecule in the reaction. Since the reaction parameters reflect the differences between these states, information on hydrogen bonding and hydrophobic interaction schemes of the liganded and unliganded macromolecule alone is principally insufficient.

Animals↗
PubMed1980

Rate enhancement of reconstitution of glyceraldehyde-3-phosphate dehydrogenase by a covalently bound coenzyme analog.

Kinetic analysis of the in vitro reconstitution of glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] from yeast showed that both oxidized and reduced coenzyme enhance the transconformation reaction, which is rate limiting in the sequential folding-association process at high enzyme concentrations (Krebs, H., Rudolph, R. & Jaenicke, R. (1979) Eur. J. Biochem. 100, 359-364). In the present study the reconstitution of the enzyme has been analyzed after covalent modification with the coenzyme analog 3-[(3-bromoacetylpyridinio)-propyl]adenosine pyrophosphate. Reconstitution of the modified enzyme, as determined by the regain of the native tryptophan fluorescence, is found to be more then 10 times faster than refolding of the unmodified apoenzyme and more than 5 times faster than that of the unmodified holoenzyme. Various degrees of denaturation and the presence of up to 0.4 M guanidine . HCl do not affect the rate of reconstitution of the modified enzyme. The kinetic effect of free or covalently bound coenzyme is discussed in terms of a decrease in free energy of the native or native-like structure or in terms of a decreased activation energy of rate-limiting steps in the process of reconstitution. Stabilization of the dimeric intermediate or acceleration of its transformation seems to be the most likely explanation for the observed effect of free or covalently bound coenzyme on the rate of reconstitution.

Adenosine Diphosphate↗
PubMed1979

Identification of the amino acid residue modified in Bacillus stearothermophilus alcohol dehydrogenase by the NAD+ analogue 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine.

4-(3-Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl-14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme-coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C-labelled coenzyme analogue and subseqeuntly carboxymethylated with unlabelled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys-38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analogue. This residue is homologous to Cys-43 in yeast alcohol dehydrogenase and Cys-46 in the horse liver enzyme but, unlike the latter two, Cys-38 is not reactive towards iodoacetate in the native bacterial enzyme.

Adenosine Diphosphate↗
PubMed1979

Affinity labelling of yeast and liver alcohol dehydrogenases with the NAD analogue 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine.

The NAD analogue 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine inactivates alcohol dehydrogenases from horse liver and yeast by modification of amino acid side chains at the active sites of the proteins. In the presence of excess inactivator the reaction is pseudo first order. The stoichiometry is one male inactivator incorporated per mole enzyme subunit. The liver enzyme is inactivated by ketoalkylation of the essential cysteine residue at position 46. No intermediate reactions of other residues are detected, and added cysteine does not influence the modification. In contrast, the labelling results with the yeast enzyme depend on cysteine treatment. The only radioactive peptide isolated is labelled on the essential cysteine residue 43.

Affinity Labels↗