Christoph Woenckhaus

Evaluation of N1-(ω-bromoacetamidoalkyl)nicotinamides as inhibitors of dehydrogenases

Abstract N 1 -(2-Bromoacetamidoethyl)nicotinamide (BAEN) and its propyl and butyl homologs were synthesized and evaluated as specific inhibitors of dehydrogenases. BAEN was a competitive inhibitor (with NAD) of yeast alcohol dehydrogenase (YADH), K I = 0.05 m . The inhibitor also irreversibly inactivated the enzyme, apparently by first forming a complex with it and then a covalent bond: YADH + BAEN YADH ⋯ BAEN inactive YADH. In the absence of ADP, K was found to be 1.3 m and K 2 was 8 min −1 . In the presence of ADP, K was 0.11 m and K 2 was 3.0 min −1 . Thus it appears that ADP promotes the binding of BAEN at a site which is near an essential group on the enzyme. ADP increased the rate of inactivation of the dehydrogenase by BAEN, but slightly decreased inactivation by bromoacetamide. A cysteinyl SH group was alkylated by BAEN in the inactive enzyme. These data suggest that there is an essential SH group near the nicotinamide binding site on the enzyme. In contrast to the yeast enzyme, mammalian dehydrogenases (horse liver alcohol, pig heart lactate, and beef liver glutamate dehydrogenases) were not competitively inhibited by BAEN (10 m m ). These enzymes were irreversibly inactivated faster by bromoacetamide than by BAEN, and ADP did not accelerate the BAEN reaction.

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.

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.

Preparation of an affinity chromatographic system for the separation of ADP binding proteins.

Abstract [4-(3-Bromoacetylpyridinio)-butyl]adenosine pyrophosphate as a structural analog of NAD+ reacts covalently with the sulfhydryl groups of thiopropyl agarose. 10-20 μmol can be bound to 1 ml gel. Stabilization of the insoluble coenzym e is attained by treatment with sodium boro hydride (NaBH4). This complex when applied to column chromatography, allow s the separation of various dehydrogenases as a result of their different complex stability coefficients. Alcohol dehydrogenase from liver, lactate dehydrogenase, and adenylate kinase, which all bind to the ADP-analog residues of the gel matrix, can thus be separated by different salt gradients. Alcohol dehydrogenase from yeast, however, does not form a complex and can easily be eluted from the column with phosphate buffer. Glyceraldehyde-3 phosphate and aldehyde dehydrogenases can be eluted by the addition of NAD+ or NADH to the buffer. The uncharged 1,4-dihydropyridin ring of the reduced coenzyme produces a more stable complex with the dehydrogenases than the oxidized form.