Felinine stability in the presence of selected urine compounds.

The stability of felinine, an amino acid present in feline urine, was investigated. Synthetic felinine was unstable in the urine of a selection of mammals. Felinine was found to stable in feline urine in which urea had been degraded. Synthetic felinine was found to react specifically with urea and did not react with urea analogues such as biuret or thiourea or other nucleophilic compounds such as ammonia which is more nucleophilic or acetamide and water which are less nucleophilic than urea. The reaction of urea and felinine was independent of pH over the range of 3-10. Urea did not react with N-acetyl-felinine suggesting a felinine N-terminal interaction with urea. Mass spectral analysis of the reaction products showed the presence of carbamylated felinine and fragmentation ions derived from carbamyl-felinine. The physiological relevance of felinine carbamylation is yet to be determined.

Interaction of sheep liver cytosolic aldehyde dehydrogenase with quercetin, resveratrol and diethylstilbestrol.

The effects of quercetin and resveratrol (substances found in red wine) on the activity of cytosolic aldehyde dehydrogenase in vitro are compared with those of the synthetic hormone diethylstilbestrol. It is proposed that quercetin inhibits the enzyme by binding competitively in both the aldehyde substrate binding-pocket and the NAD(+)-binding site, whereas resveratrol and diethylstilbestrol can only bind in the aldehyde site. When inhibition is overcome by high aldehyde and NAD(+) concentrations (1 mM of each), the modifiers enhance the activity of the enzyme; we hypothesise that this occurs through binding to the enzyme-NADH complex and consequent acceleration of the rate of dissociation of NADH. The proposed ability of quercetin to bind in both enzyme sites is supported by gel filtration experiments with and without NAD(+), by studies of the esterase activity of the enzyme, and by modelling the quercetin molecule into the known three-dimensional structure of the enzyme. The possibility that interaction between aldehyde dehydrogenase and quercetin may be of physiological significance is discussed.

The effect of quercetin, a widely distributed flavonoid in food and drink, on cytosolic aldehyde dehydrogenase: a comparison with the effect of diethylstilboestrol.

Quercetin is a flavonoid found in red wine and many other dietary sources. Observations concerning the state of ionisation and the stability of the compound over a range of pH are presented. Quercetin is a potent inhibitor of cytosolic aldehyde dehydrogenase at physiological pH when the concentration of either the substrate or the cofactor is relatively low, but it has an activatory effect when the concentrations of substrate and cofactor are both high (1 mM). Gel filtration experiments show that quercetin binds very tightly to the enzyme under conditions where the compound is neutral and when it is ionised. The binding is less in the presence of NAD(+). Quercetin cuts down the ability of the resorufin anion to bind to the enzyme. The observations are explained by a model in which quercetin binds competitively to both the coenzyme-binding site and the aldehyde-binding site; binding in the latter location, when the enzyme is in the form of the E-NADH complex, accounts for the activation. The effects of quercetin are significantly different in some respects from those of diethylstilboestrol; this is explained by the latter being able to bind to the aldehyde site but not the NAD(+) site. The possibility that quercetin may affect aldehyde dehydrogenase in vivo is discussed.

Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases.

BACKGROUND: . Enzymes of the aldehyde dehydrogenase family are required for the clearance of potentially toxic aldehydes, and are essential for the production of key metabolic regulators. The cytosolic, or class 1, aldehyde dehydrogenase (ALDH1) of higher vertebrates has an enhanced specificity for all-trans retinal, oxidising it to the powerful differentiation factor all-trans retinoic acid. Thus, ALDH1 is very likely to have a key role in vertebrate development. RESULTS: . The three-dimensional structure of sheep ALDH1 has been determined by X-ray crystallography to 2.35 A resolution. The overall tertiary and quaternary structures are very similar to those of bovine mitochondrial ALDH (ALDH2), but there are important differences in the entrance tunnel for the substrate. In the ALDH1 structure, the sidechain of the general base Glu268 is disordered and the NAD+ cofactor binds in two distinct modes. CONCLUSIONS: . The submicromolar Km of ALDH1 for all-trans retinal, and its 600-fold enhanced affinity for retinal compared to acetaldehyde, are explained by the size and shape of the substrate entrance tunnel in ALDH1. All-trans retinal fits into the active-site pocket of ALDH1, but not into the pocket of ALDH2. Two helices and one surface loop that line the tunnel are likely to have a key role in defining substrate specificity in the wider ALDH family. The relative sizes of the tunnels also suggest why the bulky alcohol aversive drug disulfiram reacts more rapidly with ALDH1 than ALDH2. The disorder of Glu268 and the observation that NAD+ binds in two distinct modes indicate that flexibility is a key facet of the enzyme reaction mechanism.

Studies on the chymotrypsin-catalysed hydrolysis of resorufin acetate and resorufin bromoacetate.

Resorufin bromoacetate is a substrate that is rapidly hydrolysed by chymotrypsin. The reaction shows a pre-steady-state burst phase that may be observed by stopped flow spectrophotometry if precautions are taken against spontaneous hydrolysis of the substrate. The strongly activating effect that the presence of the bromine atom has on the adjacent carbonyl group is reflected in the relative sizes of the kcat values for resorufin bromoacetate and resorufin acetate (e.g., 740 to 1, at pH 6) and the burst rate constants (e.g., 350 to 1, at pH 7 using 0.1 mM substrate). The pH-dependence of kcat for both substrates shows the involvement of an enzymic group of pKa about 7. With resorufin bromoacetate, a burst and a steady-state rate are still observable at pH 3.0. Unlike the case with aldehyde dehydrogenase, resorufin bromoacetate does not act as an inactivator of chymotrypsin and there is little or no incorporation of covalently-linked label when chymotrypsin and resorufin bromoacetate are incubated together. The different modes of behaviour of the two enzymes are attributable to the 'hard' or 'soft' character of the attacking enzymic nucleophilic groups.

Studies of the esterase activity of cytosolic aldehyde dehydrogenase with resorufin acetate as substrate.

Resorufin acetate is a very good substrate for sheep liver cytosolic aldehyde dehydrogenase, both from the point of view of practical spectrophotometry and in terms of information provided about the nature of the catalysis shown by this enzyme. p-Nitrophenyl (PNP) acetate competes against resorufin acetate for the enzyme's active site (although relatively weakly as the latter substrate has the lower Michaelis constant), but acetaldehyde (in the presence of NAD+) inhibits the hydrolysis of resorufin acetate only at very high aldehyde concentration. In the absence of cofactor, the rate-limiting step in the hydrolysis of resorufin acetate and of PNP acetate is hydrolysis of the common acetyl-enzyme, as shown by the observation of bursts of chromophoric product and very similar values of kcat. In the presence of NAD+ or NADH, however, the deacylation step with resorufin acetate is greatly accelerated until acylation seems to become rate-limiting, because no burst is seen under these conditions. Millimolar concentrations of Mg2+ activate the hydrolyis of resorufin acetate both in the presence and absence of cofactors. With both Mg2+ and cofactor the kcat for hydrolysis of resorufin acetate is 30-35 s-1; this is three orders of magnitude higher than the kcat for aldehyde oxidation in the presence of Mg2+, showing that the enzyme's potential catalytic efficency is very much hampered by the slowness with which NADH dissociates from its binding site. The pH profile for the hydrolysis of resorufin acetate in the presence of NAD+ or NADH fits well to a theoretical ionization curve of pKa approx. 8.2; it is suggested that this might belong to the enzyme's putative catalytic residue (Cys-302).

A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase.

1. p-Nitrophenyl (PNP) acetate and propionate show a burst of p-nitrophenoxide release when their hydrolysis is catalysed by sheep liver cytosolic aldehyde dehydrogenase. This is not seen in the presence of NAD+ or NADH, implying a change in ratedetermining step. 2. 6-Nitrodihydrocoumarin (6-NDC) shows no burst of absorbance in the visible region. We propose that the pKa of the transient "reporter group' produced during the hydrolysis of this lactone is high (approx. 10) and that the incipient covalently linked p-nitrophenoxide moiety is protonated immediately on formation. The small burst seen in the hydrolysis of 5-nitro-2-coumaranone (5-NC) suggests that the pKa of its reporter group is about 8.5. 3. NADH markedly enhances the steady-state rate with the lactones. 5-NC shows a large rapid burst of colour development in the presence of NADH; this implies that NADH decreases the pKa of the reporter group to 7-7.5. 4. In the presence of NAD+, 5-NC and 6-NDC give an unusual "negative burst' in the stopped-flow traces. We propose that, under these circumstances, acylation of the enzyme is extremely fast and that the first event seen in the stopped-flow traces is protonation of the reporter group. NAD+ also greatly increases the steady-state rate. 5. With the lactones in the presence of NADH, the kcat value (nearly 6 s-1), a measure of the deacylation rate, is compatible with the single-site model for dehydrogenase and esterase activities.

Crystallization and preliminary X-ray diffraction studies on cytosolic (class 1) aldehyde dehydrogenase from sheep liver.

The cytosolic (Class 1) aldehyde dehydrogenase (AlDH) from sheep liver has been crystallized in a form suitable for X-ray diffraction studies. The crystals, grown by vapour diffusion using 6.5 to 7.5% methoxypolyethylene glycol 5000 as precipitant, at pH 6.5, are orthorhombic with cell dimensions a = 80.7, b = 92.5, c = 151.6 A, space-group P2(1)2(1)2(1), and one dimer in the asymmetric unit. The crystals diffract to at least 2.8 A resolution. Although unmodified AlDH crystallized readily, a key factor in obtaining diffraction-quality crystals was the covalent attachment of an active site reporter group, provided by 3,4-dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one.

Probing the active site of cytoplasmic aldehyde dehydrogenase with a chromophoric reporter group.

3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one ('DMNB') reacts with cytoplasmic aldehyde dehydrogenase in a similar way to that previously observed with the structurally related p-nitrophenyl dimethylcarbamate, but provides a covalently linked p-nitrophenol-containing reporter group at the enzyme's active site. The pKa of the enzyme-linked reporter group is much higher than that of free p-nitrophenol, which is consistent with its being in a very hydrophobic environment, or possibly one containing negative charge. Upon binding of NAD+ to the modified enzyme, the pKa falls dramatically, by about 4 1/2 pH units. This implies that under these conditions there is a positive charge near the p-nitrophenoxide moiety, perhaps that of the nicotinamide ring of NAD+. The modified enzyme binds NAD+ very tightly; neither gel filtration nor dialysis is effective in separating them. However, the reporter group provides a convenient way of monitoring the displacement of this bound NAD+ when NADH is added.

Effect of some thiocarbamate compounds on aldehyde dehydrogenase and implications for the disulfiram ethanol reaction.

The effects of S-methyl diethyldithiocarbamate, S-methyl diethylmonothiocarbamate and bis(diethylcarbamoyl) disulphide on sheep liver cytoplasmic aldehyde dehydrogenase were investigated in vitro. The first compound has negligible effect. The second one is a weak inhibitor of the esterase activity of the enzyme and a weaker inhibitor of the dehydrogenase activity. A very low concentration of the third compound, however, acts as a potent inactivator of aldehyde dehydrogenase, similar in this respect to disulfiram, although somewhat slower to react. The possible involvement of these compounds in the physiological phenomenon known as the disulfiram ethanol reaction is discussed.