Kinetics of Sheep‐Liver Cytoplasmic Aldehyde Dehydrogenase

Abstract

1. Sheep liver cytoplasmic aldehyde dehydrogenase showed little pH dependence of V or k,,,. Some buffer anion effects were noted.2. The oxidation of aldehydes at pH 7.6 was quantitative but irreversible. The initial velocity data indicated a sequential mechanism for the addition of substrates. Inhibition by NADH and the product analogue 2-bromo-2-phenylacetic acid, together with the known tight binding of NADH to the free enzyme, indicated an ordered mechanism with NAD+ as leading substrate.3 . Values for the rate of binding and dissociation of NAD+ were obtained from the steady-state data. The values obtained were virtually identical with those which could be calculated from the data for the horse liver cytoplasmic enzyme. Close similarities are in general apparent for the horse and sheep liver cytoplasmic enzymes and with other tissue aldehyde dehydrogenases.4. Apparent substrate activation was observed with high concentrations of both acetaldehyde and propionaldehyde, a limiting value of 0.25 spl being obtained for k,,,. No isotope effect was observed on V using [l-'Hlpropionaldehyde as substrate suggesting that NADH release might be rate-limiting in the steady-state.5. The implications of the non-linear steady-state behaviour are discussed.In recent years aldehyde dehydrogenases from various sources have been purified to homogeneity [1,2]. More recently the purification of sheep liver cytoplasmic and mitochondrial enzymes has been reported [3] and the two major isozymes of aldehyde dehydrogenase from horse liver, which also appear to be cytoplasmic and mitochondrial in origin, have been purified and characterised [4]. An enzyme from human liver has also been studied [ 5 ] and the kinetics of aldehyde oxidation investigated, although its cellular localization is unknown.All the kinetic investigations so far reported suggest sequential mechanisms but the order of substrate addition varies from one enzyme to another. Pig brain, horse liver and bovine liver enzymes have been reported to follow compulsory order mechanisms in which NAD+ binds before aldehyde [2,6-8], but evidence has also been presented for the yeast enzyme [9] which indicates an ordered mechanism in which aldehyde binds first.The human liver enzyme appears to be partially random in its addition of its substrates, although most of the reaction is considered to proceed by means of an initial binary NAD' . enzyme complex [5]. In view of the importance of these enzymes in an understanding of human alcohol metabolism we have undertaken a detailed kinetic study of sheep liver cytoplasmic aldehyde dehydrogenase in order to determine the steady-state parameters for the reaction. MATERIALS AND METHODS MaterialsNADH (grade 111) and NAD' (grade 111) were obtained from Sigma Chemical Co. (St Louis, Mo., U.S.A.). The NADH was obtained as preweighed vials and the concentration was checked by absorbance measurements at 340 nm assuming a molar absorption coefficient (8) of 6 . 2 2~ lo3 M-l cm-I [lo]. NADt was used without further purification since no detectable...

Evidence for two-step binding of reduced nicotinamide-adenine dinucleotide to aldehyde dehydrogenase

“…The enzyme concentrations quoted represent the concentration of NADH-binding sites determined from a fluorimetric titration similar to that described by Luisi et al (1973), based on the enhancement of nucleotide fluorescence (approx. 5.6-fold) which results when NADH binds to the enzyme (MacGibbon, 1976). Plots of 1/(1-R) versus [NADH]/R, where R represents the fraction of occupied binding sites, were linear, showing no evidence for subunit interactions and giving a value of 1.2AM for the E-NADH dissociation constant, similar to that determined from steadystate-inhibition data (MacGibbon etal., 1977).…”

“…[E]O) values found for high concentrations of propionaldehyde and acetaldehyde in the steady state, of 0.25 and 0.27 s-respectively (MacGibbon et al, 1977). Thus for sheep liver cytoplasmic aldehyde dehydrogenase, as was reported for the Fl isoenzyme of horse liver (Eckfeldt & Yonetani, 1976) the slow rate of NADH release contributes significantly to the limitation of enzyme catalytic-centre velocity, a result that may prove to be general for all tissue aldehyde dehydrogenases.…”

“…Although with pyridine-3-aldehydeadenine dinucleotide a significantly different slower process was observed, this coenzyme analogue did not act as a coenzyme with propionaldehyde in the steady-state reaction (although it did act as an aldehyde substrate with NAD+ as coenzyme), and may not be suitable as a displacing agent (MacGibbon, 1976).…”

“…We could include the appropriate fluorescenceenhancement factors (MacGibbon, 1976) and determine the relative amplitudes of the two phases directly from the computer print-out of the logarithm of the difference of the fluorescence as a function of time for the particular model being considered.…”

Changes in the fluorescence of bound nucleotide during the reaction catalysed by glucose‐fructose oxidoreductase from Zymomonas mobilis

“…Rapid changes in nucleotide fluorescence were observed using excitation at 340 nm and emission through Wratten 47B and 2B filters which have maximum transmission at 435 nm [8]. All experiments were carried out in 10 mM K/Mes pH 6.5, containing 30 mM NaCl and 2 mM MgC12 at 25"C, except for an experiment repeated at 21 C for comparison with our previous absorbance experiment [ 31.…”