scholarly journals Multiple binding of thallium and rubidium to potassium-activated yeast aldehyde dehydrogenase. Influences on tertiary structure, stability and catalytic activity

1982 ◽  
Vol 207 (1) ◽  
pp. 73-80 ◽  
Author(s):  
K A Bostian ◽  
G F Betts ◽  
W K Man ◽  
M N Hughes
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Binding Sites ◽  
Tertiary Structure ◽  
Equilibrium Dialysis ◽  
Structure Stability ◽  
Conformation Changes ◽  
Steady State Kinetic ◽  
Multiple Binding ◽  
Inhibitory Site

Univalent cation activators of aldehyde dehydrogenase have dual effects, both interpreted as cation-induced or -stabilized conformation changes. These two processes are differentiated by the time scales of their associated changes in activity. Using Tl+ as an activator, under certain conditions, the slower change in activity saturates at a Tl+ concentration which is only 0.1 Ks for the faster change. This, together with evidence for cation-induced rather than cation-stabilized conformation changes, is used to propose separate binding sites for cations responsible for the two activation processes. Equilibrium dialysis indicates 4 binding sites per active site for Rb+ or 6 sites for Tl+. At least one of the additional sites for Tl+ is an inhibitory site which has been differentiated from the activator sites on the basis of steady-state and pre-steady-state kinetic data.

scholarly journals Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

2000 ◽  
Vol 352 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Roberto VELASCO-GARCÍA ◽  
Lilian GONZÁLEZ-SEGURA ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Metabolic Role ◽  
Steady State Kinetic ◽  
State Kinetic

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)+ to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD+- and NADP+-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)+ and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)+ binds first and NAD(P)H leaves last, particularly in the NADP+-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP+-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose.

scholarly journals Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme

1978 ◽  
Vol 171 (3) ◽  
pp. 527-531 ◽  
Author(s):  
A K H MacGibbon ◽  
L F Blackwell ◽  
P D Buckley
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Kinetic Properties ◽  
Protein Fluorescence ◽  
Sheep Liver ◽  
Steady State Kinetic ◽  
Wide Range ◽  
Liver Aldehyde Dehydrogenase

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 × 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.

scholarly journals Bovine lens aldehyde dehydrogenase. Kinetics and mechanism

1983 ◽  
Vol 215 (2) ◽  
pp. 361-368 ◽  
Author(s):  
H H Ting ◽  
M J C Crabbe
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Ternary Complex ◽  
Succinic Semialdehyde ◽  
Complex Mechanism ◽  
Thiol Groups ◽  
Bovine Lens ◽  
Steady State Kinetic ◽  
State Kinetic

Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.

scholarly journals Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes

1992 ◽  
Vol 287 (1) ◽  
pp. 145-150 ◽  
Author(s):  
G T M Henehan ◽  
K F Tipton
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Human Erythrocytes ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Sequential Mechanism ◽  
Kinetics Of

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear ‘mixed inhibition’ pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.

scholarly journals Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase

1977 ◽  
Vol 167 (2) ◽  
pp. 469-477 ◽  
Author(s):  
Alastair K. H. MacGibbon ◽  
Leonard F. Blackwell ◽  
Paul D. Buckley
Keyword(s):  
Steady State ◽  
Isotope Effect ◽  
Aldehyde Dehydrogenase ◽  
Rate Constant ◽  
Kinetic Studies ◽  
Similar Data ◽  
Protein Fluorescence ◽  
Sheep Liver ◽  
Steady State Kinetic ◽  
Liver Aldehyde Dehydrogenase

Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD+ and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD+ and propionaldehyde with the enzyme were varied were fitted to the following mechanism: [Formula: see text] where the release of NADH is slow. By monitoring the quenching of protein fluorescence on the binding of NAD+, estimates of 2×105 litre·mol−1·s−1 and 2s−1 were obtained for k+1 and k−1 respectively. Although k+3 could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s−1, k+2 and k−2 could not be determined uniquely, but could be related by the equation: (k−2+k+3)/k+2 =50×10−6mol·litre−1. No significant isotope effect was observed when [1-2H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k−2+k+3)/k+2=2.3×10 −3mol·litre−1 and k+3 is 23s−1. When [1,2,2,2-2H]acetaldehyde was used, no isotope effect was observed on k+3, but there was a significant effect on k+2 and k−2. A burst of NADH production has also been observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.

Reversibly Sampling Conformations and Binding Modes Using Molecular Darting

2020 ◽  
Author(s):  
Samuel C. Gill ◽  
David Mobley
Keyword(s):  
Monte Carlo ◽  
Ligand Binding ◽  
Binding Sites ◽  
Degrees Of Freedom ◽  
Dynamics Simulation ◽  
Hiv Integrase ◽  
Binding Modes ◽  
System A ◽  
Multiple Binding ◽  
Internal Degrees Of Freedom

<div>Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.</div><div>We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems.</div>

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