Mechanism of a long-chain fatty aldehyde dehydrogenase induced during the development of bioluminescence in Beneckea harveyi

1983 ◽  
Vol 61 (5) ◽  
pp. 301-306 ◽  
Author(s):  
Andrew Bognar ◽  
Edward Meighen
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Fatty Aldehyde ◽  
Rate Limiting Step ◽  
Dead End ◽  
Sequential Mechanism ◽  
High Concentrations ◽  
Substrate Protection ◽  
Long Chain

The kinetic mechanism of a long-chain aldehyde dehydrogenase that is induced during the development of bioluminescence in Beneckea harveyi has been investigated. Parallel lines were obtained in Lineweaver–Burk plots with NAD+ and long-chain aldehydes (heptaldehyde, nonylaldehyde). However, product and dead-end inhibitor studies, substrate protection (NAD+, aldehyde) against inactivation with N-ethylmaleimide, and in particular, a noncompetitive substrate inhibition pattern with aldehyde at high concentrations showed that aldehyde dehydrogenase had a sequential mechanism. The data were consistent with a nonrapid equilibrium random mechanism with a preferred order of addition of substrates (NAD+, aldehyde) and an ordered release of products with NADH release being the last and rate-limiting step in the reaction, a mechanism very similar to that found for short-chain mammalian aldehyde dehydrogenases. The present experiments emphasize the caution that must be taken in interpreting parallel patterns in initial velocity experiments, as well as the difficulty in classifying sequential enzyme mechanisms as either strictly ordered or random.

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 Kinetic mechanism of the dimeric ATP sulfurylase from plants

2013 ◽  
Vol 33 (4) ◽  
Author(s):  
Geoffrey E. Ravilious ◽  
Jonathan Herrmann ◽  
Soon Goo Lee ◽  
Corey S. Westfall ◽  
Joseph M. Jez
Keyword(s):  
Initial Velocity ◽  
Competitive Inhibition ◽  
Tight Binding ◽  
Atp Synthesis ◽  
Kinetic Mechanism ◽  
Titration Calorimetry ◽  
Atp Sulfurylase ◽  
Dead End ◽  
Sequential Mechanism ◽  
Enzyme Functions

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.

Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions.

1976 ◽  
Vol 22 (4) ◽  
pp. 417-421 ◽  
Author(s):  
J H Stromme ◽  
L Theodorsen
Keyword(s):  
Clinical Chemistry ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Reaction Conditions ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Dead End ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Assay Conditions

Abstract Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively.

scholarly journals Human glutamic-γ-semialdehyde dehydrogenase. Kinetic mechanism

1989 ◽  
Vol 261 (3) ◽  
pp. 935-943 ◽  
Author(s):  
C Forte-McRobbie ◽  
R Pietruszko
Keyword(s):  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Maximal Velocity ◽  
Semialdehyde Dehydrogenase ◽  
Rate Limiting Step ◽  
Dead End ◽  
Inhibition Studies ◽  
Rate Limiting ◽  
Competitive Pattern ◽  
Pattern Versus

The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.

scholarly journals γ-Glutamyltranspeptidase-catalysed acyl-transfer to the added acceptor does not proceed via the ping-pong mechanism

1994 ◽  
Vol 304 (3) ◽  
pp. 869-876 ◽  
Author(s):  
M Y Gololobov ◽  
R C Bateman
Keyword(s):  
Serine Proteases ◽  
Kinetic Mechanism ◽  
Inhibitory Effect ◽  
Acyl Transfer ◽  
Transfer Reactions ◽  
Acceptor Complex ◽  
Dead End ◽  
Sequential Mechanism ◽  
Donor Acceptor ◽  
Acyl Transfer Reactions

Acyl-transfer catalysed by gamma-glutamyltranspeptidase from bovine kidney was studied using gamma-L- and gamma-D-Glu-p-nitroanilide as the donor and GlyGly as the acceptor. The transfer of the gamma-Glu group to GlyGly was shown to be accompanied by transfer of the gamma-Glu group to water (hydrolysis). The results were compared with acyl-transfer catalysed by the representative serine protease, alpha-chymotrypsin. The main difference between the kinetic mechanism of the acyl-transfer reactions catalysed by these enzymes, which contain an active-site serine and form an acyl-enzyme intermediate but belong to different enzyme classes, was found to consist in the role of the enzyme-donor-acceptor complex. This complex is not formed at any acceptor concentrations in the acyl-transfer reactions catalysed by the serine proteases. In contrast, in the gamma-glutamyltranspeptidase-catalysed acyl-transfer the pathway going through the ternary enzyme-donor-acceptor complex formed from the enzyme-acceptor complex becomes the main pathway of the transfer reaction even at moderate acceptor concentrations. As a result, gamma-glutamyltranspeptidase catalysis follows a sequential mechanism with random equilibrium addition of the substrates and ordered release of the products. The second distinction concerns the inhibitory effect of the acceptor. In the case of alpha-chymotrypsin this was the result of true inhibition, i.e. a dead-end formation of the enzyme-acceptor complex. A salt effect caused by the acceptor was the rationale of a similar effect observed in acyl-transfer catalysed by gamma-glutamyltranspeptidase.

Impaired skin barrier function due to reduced ω- O -acylceramide levels in a mouse model of Sjögren–Larsson syndrome

2021 ◽  
Author(s):  
Koki Nojiri ◽  
Shuhei Fudetani ◽  
Ayami Arai ◽  
Takuya Kitamura ◽  
Takayuki Sassa ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Aldehyde Dehydrogenase ◽  
Barrier Function ◽  
Skin Barrier ◽  
Skin Barrier Function ◽  
Fatty Aldehyde ◽  
Chain Base ◽  
Long Chain Base ◽  
Larsson Syndrome ◽  
Long Chain

Sjögren–Larsson syndrome (SLS) is an inherited neurocutaneous disorder whose causative gene encodes the fatty aldehyde dehydrogenase ALDH3A2. To date, the detailed molecular mechanism of the skin pathology of SLS has remained largely unclear. We generated double knockout (DKO) mice for Aldh3a2 and its homolog Aldh3b2 (a pseudogene in humans). These mice showed hyperkeratosis and reduced fatty aldehyde dehydrogenase activity and skin barrier function. The levels of ω- O -acylceramides (acylceramides), which are specialized ceramides essential for skin barrier function, in the epidermis of DKO mice were about 60% of those in wild type mice. In the DKO mice, levels of acylceramide precursors (ω-hydroxy ceramides and triglycerides) were increased, suggesting that the final step of acylceramide production was inhibited. A decrease in acylceramide levels was also observed in human immortalized keratinocytes lacking ALDH3A2 . Differentiated keratinocytes prepared from the DKO mice exhibited impaired long-chain base metabolism. Based on these results, we propose that the long-chain-base–derived fatty aldehydes that accumulate in DKO mice and SLS patients attack and inhibit the enzyme involved in the final step of acylceramide. Our findings provide insight into the pathogenesis of the skin symptoms of SLS, i.e., decreased acylceramide production, and its molecular mechanism.

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.

Circulating Ceramides and Sphingomyelins and Risk of Mortality: The Cardiovascular Health Study

2021 ◽  
Author(s):  
Amanda M Fretts ◽  
Paul N Jensen ◽  
Andrew N Hoofnagle ◽  
Barbara McKnight ◽  
Colleen M Sitlani ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cardiovascular Health ◽  
Health Study ◽  
Cardiovascular Health Study ◽  
Risk Of Death ◽  
Risk Of Mortality ◽  
Increased Risk ◽  
High Concentrations ◽  
Long Chain

Abstract Background Recent studies suggest that associations of ceramides (Cer) and sphingomyelins (SM) with health outcomes differ according to the fatty acid acylated to the sphingoid backbone. The purpose of this study was to assess associations of Cer and SM species with mortality. Methods The study population included participants from the Cardiovascular Health Study (CHS), a community-based cohort of adults aged ≥65 years who were followed from 1992–2015 (n = 4612). Associations of plasma Cer and SM species carrying long-chain (i.e., 16:0) and very-long-chain (i.e., 20:0, 22:0, 24:0) saturated fatty acids with mortality were assessed using Cox proportional hazards models. Results During a median follow-up of 10.2 years, 4099 deaths occurred. High concentrations of Cer and SM carrying fatty acid 16:0 were each associated with an increased risk of mortality. Conversely, high concentrations of several ceramide and sphingomyelin species carrying longer fatty acids were each associated with a decreased risk of mortality. The hazard ratios for total mortality per 2-fold difference in each Cer and SM species were: 1.89 (95% CI), 1.65–2.17 for Cer-16, 0.79 (95% CI, 0.70–0.88) for Cer-22, 0.74 (95% CI, 0.65–0.84) for Cer-24, 2.51 (95% CI, 2.01–3.14) for SM-16, 0.68 (95% CI, 0.58–0.79) for SM-20, 0.57 (95% CI, 0.49–0.67) for SM-22, and 0.66 (0.57–0.75) for SM-24. We found no association of Cer-20 with risk of death. Conclusions Associations of Cer and SM with the risk of death differ according to the length of their acylated saturated fatty acid. Future studies are needed to explore mechanisms underlying these relationships.

scholarly journals The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker's yeast

1972 ◽  
Vol 126 (1) ◽  
pp. 35-47 ◽  
Author(s):  
B. Middleton
Keyword(s):  
Ph Value ◽  
Acyl Chain ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Hydroxyl Group ◽  
Acetyl Coa ◽  
Hydrophobic Region ◽  
Active Centre ◽  
Acyl Chain Length ◽  
Inhibition Pattern

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have Ki values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of Ki with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).

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