Biomedical subjects
Publications and source records attributed to J F Biellmann.
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The kinetic properties of 18 NAD+ analogues, with alterations to the nicotinamide moiety, have been studied with respect to dogfish, M4, rabbit M4 and beef H4 lactate dehydrogenase. The size of the groups present at C-3 of the pyridinium can be increased quite extensively without loss of coenzyme activity. Groups tested were thioamide, methyl, ethyl, diazoketone and chloroacetyl. Substitutions at positions C-4 and C-5 prevent proper positioning for hydride transfer and can hinder binding to the enzyme. The kinetic properties of pyridine-adenine dinucleotide and its 3-iodo derivative reveal the binding role of the amide at C-3 whereas 3-cyanopyridine-adenine dinucleotide is a strong inhibitor.
Aspartate-beta-semialdehyde dehydrogenase, from an Escherichia coli mutant derepressed for the biosynthesis of L-lysine, has been purified to homogeneity. Its isoelectric point is pH 4.3. This enzyme has a molecular weight of 77000 and is composed of two identical or highly similar subunits of molecular weight 38000 +/- 2000. Their N-terminal amino-acid sequence is Met-Lys-Asx-Val-Gly-. Three cysteine residues per subunit were detected: two are reactive in the native enzyme and one is partially protected by the substrate. Formation of an acyl-enzyme intermediate was also detected. Correlation of the 1H nucleár magnetic resonance spectrum of [4-2H]NADPH produced from [4-2H]NADP+ indicated that aspartate beta-semialdehyde dehydrogenase transfers the pro-S hydrogen from NADPH (class B dehydrogenase). A short comparison with the corresponding yeast enzyme is given.
The substrate binding site of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli was studied by affinity labeling with L-2-amino-4-oxo-5-chloropentanoic acid. The substrate analogue irreversibly inactivates the enzyme with pseudo-first-order kinetics and with a half-of-the-sites reactivity. The substrate aspartate beta-semialdehyde protects the enzyme against the inactivation. A single group is labeled at the active site and is concluded to be the side-chain of a histidine residue. The amino acid sequence around the active site residue was established from a peptic digest of the labeled enzyme: Phe-Val-Gly-Gly-Asp-(modified residue)-Thr-Val-Ser.
3-Chloroacetylpyridine-adenine dinucleotide phosphate (clac3PdADP+, a NADP+ alkylating analogue, irreversibly inactivates aspartate-beta-semialdehyde dehydrogenase with pseudo-first-order kinetics. NADP+ and NADPH, but not the substrate, protected the enzyme from inactivation. The pH dependence of the inactivation kinetics was determined. Incorporation of 1 mol cl[14C]-ac3PdADP+/dimer totally inactivates the enzyme. Successive alkylation by the coenzyme analogue and by the substrate analogue, L-2-amino-4-oxo-5-chloropentanoic acid, was studied. After inactivation with the coenzyme analogue, no incorporation of the substrate analogue was detected. However, when the enzyme was first inactivated with the substrate analogue, the protein could subsequently be alkylated with the coenzyme analogue. The binding of NADP+ and NADPH to aspartate-beta-semialdehyde dehydrogenase was determined by fluorescence.
Two aldehyde reductases (EC 1.1.1.2), I and II, have been isolated from pig liver. Both are monomeric (Mr approximately equal to 35 000) and NADPH-dependent. Their activity is inhibited by barbiturates. The enzymes reduce essentially aromatic aldehydes, with a preference for those bearing an electron-withdrawing group in the para position. Substrates with a carboxyl group are specially good substrates for reductase I. This may indicate the presence of a positively charged group in the substrate binding site. The binding of NADPH to reductase I causes a red shift of the coenzyme absorption; this shift is characteristic of B-stereospecific dehydrogenases. Nevertheless, this is not confirmed by the stereochemical study with labelled NADPH. The pro-R hydrogen of NADPH is transferred to the re face of the aldehyde. The stereochemical course of reductase I is identical to that of liver alcohol dehydrogenase, but the two enzymes differ by the absence of Zn and of reactive thiol in reductase I, and by the action of pyrazole on the activity. Considerable differences in substrate specificity and immunological properties have been found between reductase I and II but reductases I from liver of different species have some relationship. Reductase I from pig brain and pig kidney seem to be identical to reductase I from pig liver.
A new procedure for the activity measurement of NAD(P)+-dependent dehydrogenases has been devised using an electron-transferring agent, phenazine methosulfate, and an electron acceptor, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide. The reduction of the latter is determined by an increase in absorbance at 578 nm. 3-Iodopyridineadenine dinucleotide was found to be active as an hydride acceptor with horse liver alcohol dehydrogenase and lactate dehydrogenase but showed no activity with glyceraldehyde-3-phosphate dehydrogenase nor did its phosphate with 3-phosphogluconate dehydrogenase.
The fate of the iodide liberated during carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase has been determined with 125I-labeled iodoacetate. The [125I]iodoacetic acid was prepared from mesyloxyacetic acid and sodium [125I]iodide. When carboxymethylation of the enzyme is carried out in solution or in the crystalline state, no iodide is bound to the protein. The rate of iodide during the reaction of iodoacetate, determined with an iodide-specific electrode, has been found to be biphasic: the fast phase corresponds to the carboxymethylation and the slow phase to iodide liberation due to the presence of protein. With 3-iodopropionate (2.5 mM), no inactivation was detected, but in the presence of the enzyme, 10 equivalents of iodide were liberated per subunit in 1 hr. NADH does not inhibit this reaction. The electron density attributed to an iodide bound to the zinc atom of the crystalline enzyme is reinterpreted in view of these results as due to an imidazole bound to the active-site zinc. In the carboxymethylation, the reactivity of bromoacetate is higher than that of iodoacetate.
3-Chloroacetylpyridine--adenine dinucleotide phosphate is both active as a hydride acceptor and inactivates estradiol 17 beta-dehydrogenase. This coenzyme analogue behaves like an affinity label. The inactivation kinetics are discussed in relation to those observed with 3-chloroacetylpyridine--adenine dinucleotide. The pH dependence of the rate of inactivation, in combination with determination of the number of reactive cysteine residues, pointed to the alkylation of one cysteine residue/subunit. The stoichiometry was one molecule of dinucleotide per subunit and no cooperativity was detected. When 14C-labeled dinucleotide was used, the 14C label was found mainly in one peptide, accounting for 90% of the incorporated radioactivity, whereas in previous work it had been shown that 3-chloroacetylpyridine--adenine dinucleotide is an affinity reagent which labels three peptides.
Two inhibitors of lactate dehydrogenase generated during NADH storage have been isolated by chromatography. One is a dimer of the dinucleotide where the AMP moiety is unmodified. The other is also generated from NAD+ in the presence of a high concentration of phosphate ions at alkaline pH. This inhibitor was proved to be the addition compound of one phosphate group to position C-4 of the nicotinamide ring of NAD+ by NMR spectroscopy, enzymatic cleavage, and dissociation to NAD+ at neutral pH. This compound is a competitive inhibitor with respect to NAD+ in the presence of the lactate dehydrogenase with a Ki of 2 X 10(-7) M. The interaction of this inhibitor with lactate dehydrogenase is discussed relative to the structure of this enzyme.
A stopped-flow spectrophotometer was modified so that the volumes to be mixed were in a ratio of 1:50. Using this instrument, we have shown that the effective substrate in the reduction of acetaldehyde catalyzed by horse liver alcohol dehydrogenase was the carbonyl form of acetaldehyde and that the enzyme does not catalyze the dehydration of the hydrated form of acetaldehyde. Unlike trifluoroacetaldehyde hydrate, which is a competitive inhibitor with respect to ethanol, acetaldehyde hydrate did not inhibit the enzymatic reaction at concentrations as high as 60 mM.
The binding of Cibacron F3GA to orthorhombic crystals of liver alcohol dehydrogenase has been studied to 0.37-nm resolution. Similarities in the binding of this dye were found for rings B, C and D with the binding of the coenzyme NAD+. However, ring A of the dye and the nicotinamide ribose part of the coenzyme are quite differently bound to the enzyme.
The analogues of the coenzyme NADP+, nicotinamide--8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine--adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was found to be active in the hydrogen transfer adn io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP+, NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+ -NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.
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The mechanism of the inactivation of 6-phosphogluconate dehydrogenase from Candida utilis with two coenzyme analogues can be differentiated on the basis of kinetic studies and of the properties of the inactivated enzyme. 3-Chloroacetylpyridine--adenine dinucleotide phosphate is clearly an affinity label and 3-choloroacetylpyridine--adenine dinucleotide a second-order reagent. For 3-chloroacetylpyridine--adenine dinucleotide phosphate, there is a loss of one thiol per subunit at complete inactivation whereas for 3-chloroacetylpyridine--adenine dinucleotide 2.7 thiol groups are lost. The fluorescence of the protein is quenched after alkylation by 3-chloroacetylpyridine--adenine dinucleotide phosphate and there is no quenching after the inactivation with 3-chloroacetylpyridine--adenine dinucleotide.
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An alkylating analogue of NADP+ the 3-chloroacetylpyridine adenine dinucleotide phosphate was prepared from 3-diazoacetylpyridine adenine dinucleotide phosphate which was obtained by enzymatic transglucosidation of NADP+. The 3-diazoacetylpyridine adenine dinucleotide phosphate proved to be more unstable when compared to the corresponding NAD+ analogue. The alkylation of several dehydrogenases using this alkylating analogue is mentioned.