Identification of lysine 74 in the pyruvate binding site of alanine dehydrogenase from Bacillus subtilis. Chemical modification with 2,4,6-trinitrobenzenesulfonic acid, n-succinimidyl 3-(2-pyridyldithio)propionate, and 5'-(p-(fluorosulfonyl)benzoyl)adenosine.

L-Alanine dehydrogenase from Bacillus subtilis was inactivated with two different lysine-directed chemical reagents, i.e. 2,4, 6-trinitrobenzenesulfonic acid and N-succinimidyl 3-(2-pyridyldithio)propionate. In both cases, the inactivation followed pseudo first-order kinetics, with a 1:1 stoichiometric ratio between the reagent and the enzyme subunits. Partial protection of the active site from inactivation could be obtained by each of the substrates, NADH or pyruvate, but complete protection could only be achieved in the presence of the ternary complex E.NADH. pyruvate. The nucleotide analogue of NADH, 5'-(p-(fluorosulfonyl)benzoyl)adenosine was also used for affinity labeling of the enzyme active site. Differential peptide mapping, performed both in the presence and in the absence of the substrates, followed by reversed phase high performance liquid chromatography separation, diode-array analysis, mass spectrometry, and N-terminal sequencing of the resulting peptides, allowed the identification of lysine 74 in the active site of the enzyme. This residue, which is conserved among all L-alanine dehydrogenases, is most likely the residue previously postulated to be necessary for the binding of pyruvate in the active site. Surprisingly, this residue and the surrounding conserved residues are not found in amino acid dehydrogenases like glutamate, leucine, phenylalanine, or valine dehydrogenases, suggesting that A-stereospecific amino acid dehydrogenases such as L-alanine dehydrogenase could have evolved apart from the B-stereospecific amino acid dehydrogenases.

Automated solid-phase synthesis of cyclic peptides bearing a side-chain tail designed for subsequent chemical grafting.

Recent developments in allyl chemistry and palladium solubilization allow automated continuous-flow solid-phase synthesis of cyclic or branched peptides, with specific side-chain cleavage and on-line cyclization. In this paper, we adapted the method to the synthesis of cyclic peptides bearing an anchoring tail on a side chain of the cycle. Side products were obtained with the standard procedure and an additional washing step had to be introduced in the synthesis protocol to remove side products resulting from the palladium allyl cleavage step. The method is illustrated by the automated synthesis of cyclo[-DVal-Arg-Gly-Asp-Glu (-epsilon Ahx-Cys-NH2)-] which contains the Arg-Gly-Asp adhesion motif (RGD) recognized by cellular integrins. The tail of the peptide was designed with a thiol at the carboxylic end to allow subsequent grafting by covalent attachment. Such tailed cyclic peptides can be grafted on different supports for new applications in biomaterial design, cell adhesion assays, affinity chromatography, immunization, vaccine development, ELISA kits, and the building of libraries of conformationally constrained peptides.

Amination of polystyrene microwells: application to the covalent grafting of DNA probes for hybridization assays.

Amine functionalization of polystyrene microwells for covalent binding of DNA is described. Polystyrene support was first carboxylated by permanganate oxidation in diluted sulfuric acid. These functions were activated with water-soluble carbodiimide and grafted with N-methyl-1,3-propane diamine to introduce a free secondary amino group on the support. The samples were characterized by X-ray photoelectron spectroscopy and radiochemical assay. The conditions for covalent linkage of secondary amine on polystyrene microwells were optimized. Functionalized supports were used for covalent binding of a DNA capture probe for the detection of human cytomegalovirus in a sandwich hybridization assay. Sensitivity of the assay compared very well with a commercially available surface, Covalink-NH, microwell plate obtained by electromagnetic irradiation.

Similarities between alanine dehydrogenase and the N-terminal part of pyridine nucleotide transhydrogenase and their possible implication in the virulence mechanism of Mycobacterium tuberculosis.

Recent developments in simultaneous multiple alignment methods of protein sequences allow prediction of structural similarity in related proteins. Alanine dehydrogenase and the N-terminal sequence of pyridine nucleotide transhydrogenase were compared for their sequences. High similarities of sequences were observed especially in their NAD(H)-binding sites. These similarities suggest that antibodies which recognized the alanine dehydrogenase of Mycobacterium tuberculosis can also be directed against the membrane bound pyridine nucleotide transhydrogenase. If this is the case, the virulent property of this pathogen could be linked to its higher synthesis of NADPH necessary for its anabolism.

Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase is a tetrameric enzyme containing zinc. Initially we confirmed the presence of two zinc atoms per subunit. Incubation of the enzyme with increasing concentrations of dithiothreitol, a method for partial chelation, allowed first the reduction of four disulphide bridges per enzyme, but eventually was sufficient to chelate the structural zinc atom without having any effect on the zinc located in the active site. The enzyme activity was not affected but the enzyme became very sensitive to heat denaturation. Chelation by EDTA was also performed. Given its location at an external position in the globular protein, protected in each subunit by one disulphide bridge, the results establish that the second zinc atom present on each enzymic subunit plays a prominent conformational role, probably by stabilizing the tertiary structure of yeast alcohol dehydrogenase. Recovery experiments were performed by incubation of the native enzyme, or the dithiothreitol-treated enzyme, with a small amount of Zn2+. A stabilization effect was found when the structural zinc was re-incorporated after its removal by dithiothreitol. In all cases a large increase in activity was also observed, which was much greater than that expected based on the amount of re-incorporated zinc atom, suggesting the re-activation of some inactive commercial enzyme which had lost some of its original catalytic zinc atoms.