Time-Dependent Effects of Trimethylamine-N-Oxide/Urea on Lactate Dehydrogenase Activity: An Unexplored Dimension of the Adaptation Paradigm

Baskakov, Ilia V.; Bolen, D. Wayne

doi:10.1016/s0006-3495(98)77971-8

Cited by 26 publications

(20 citation statements)

References 22 publications

(30 reference statements)

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“…TMAO was also shown to protect against urea-induced dissociation and inactivation of lactate dehydrogenase in vitro (32). TMAO promotes protein folding by preferential hydration of the exposed peptide backbone of an unfolded protein (33).…”

Section: Figmentioning

confidence: 98%

Natural Osmolyte Trimethylamine N-Oxide Corrects Assembly Defects of Mutant Branched-chain α-Ketoacid Decarboxylase in Maple Syrup Urine Disease

Song

Chuang

2001

Journal of Biological Chemistry

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Maple syrup urine disease is caused by deficiency in the mitochondrial branched-chain ␣-ketoacid dehydrogenase (BCKD) complex. The clinical phenotype includes often fatal ketoacidosis, neurological derangement, and mental retardation. The type IA mutations Y393N-␣, Y368C-␣, and F364C-␣, which occur in the E1␣ subunit of the decarboxylase (E1) component of the BCKD complex, impede the conversion of an ␣␤ heterodimeric intermediate to a native ␣ 2 ␤ 2 heterotetramer in the E1 assembly pathway. In the present study, we show that a natural osmolyte trimethylamine N-oxide (TMAO) at the optimal 1 M concentration restores E1 activity, up to 50% of the wild type, in the mutant E1 carrying the above missense mutations. TMAO promotes the conversion of otherwise trapped mutant heterodimers to active heterotetramers. This slow step does not involve dissociation/reassociation of the mutant heterodimers, which are preformed in the presence of chaperonins GroEL/GroES and Mg-ATP. The TMAO-stimulated mutant E1 activity is remarkably stable upon removal of the osmolyte, when cofactor thiamine pyrophosphate and the transacylase component of the BCKD complex are present. The above in vitro results offer the use of chemical chaperones such as TMAO as an approach to mitigate assembly defects caused by maple syrup urine disease mutations. Maple syrup urine disease (MSUD)1 or branched-chain ketoaciduria is an autosomal recessive metabolic disorder in the catabolism of branched-chain ␣-ketoacids derived from leucine, isoleucine, and valine (1). The accumulated branched-chain ␣-ketoacids and amino acids are secreted in the urine, giving rise to a distinct maple syrup odor and hence the name of the disease (2). Based on the clinical presentation and biochemical responses to thiamine treatment, MSUD can be grouped into five distinct phenotypes, classic, intermediate, intermittent, thiamine-responsive, and dihydrolipoamide dehydrogenase (E3)-deficient forms (1). Classic MSUD has a neonatal onset of often fatal ketoacidosis and encephalopathy, which lead to mental retardation in survivors. Intermediate and intermittent forms show milder symptoms with a late onset. Patients with thiamine-responsive MSUD respond to pharmacological doses of thiamine with returns to the normal levels of branched-chain amino acids (3). Patients with E3 deficiency have combined enzyme impairments in ␣-ketoacid dehydrogenase complexes and usually die in infancy with severe lactic acidosis (4). The prevalence of MSUD is 1 in 185,000 newborns worldwide, but in certain populations, for example the Mennonites, the incidence is as high as 1 in 176 life births, as a result of consanguinity.The enzyme affected in MSUD, the mitochondrial branchedchain ␣-ketoacid dehydrogenase (BCKD) complex, is a multienzyme complex of 4 -5 million daltons. It is organized about a 24-meric cubic core of dihydrolipoyl transacylase (E2). Attached to the E2 core are multiple copies of branched-chain ␣-ketoacid decarboxylase (E1), E3, BCKD kinase, and BCKD phosphatase (5, 6). The kinase and th...

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Section: Figmentioning

confidence: 98%

Natural Osmolyte Trimethylamine N-Oxide Corrects Assembly Defects of Mutant Branched-chain α-Ketoacid Decarboxylase in Maple Syrup Urine Disease

Song

Chuang

2001

Journal of Biological Chemistry

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“…TMAO can restore enzyme function that has been lost because of the presence of urea (6,(10)(11)(12) by restoring the protein to its native structure (13)(14)(15). The mechanism of action of these protective osmolytes is not understood fully; both direct (16)(17)(18)(19) and indirect (13,(19)(20)(21) interactions have been proposed, and the mechanism may be molecule-specific (14).…”

mentioning

confidence: 99%

Counteraction of urea-induced protein denaturation by trimethylamine N -oxide: A chemical chaperone at atomic resolution

Bennion

Daggett

2004

Proc. Natl. Acad. Sci. U.S.A.

300

287

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Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea and 4 M TMAO͞8 M urea solutions, in addition to other control simulations, to investigate this effect at the atomic level. In 8 M urea, the protein unfolds, and urea acts in both a direct and indirect manner to achieve this effect. In contrast, introduction of 4 M TMAO counteracts the effect of urea and the protein remains well structured. TMAO makes few direct interactions with the protein. Instead, it prevents unfolding of the protein by structuring the solvent. In particular, TMAO orders the solvent and discourages it from competing with intraprotein H bonds and breaking up the hydrophobic core of the protein.M echanisms have evolved in nature to allow living organisms to compensate for extreme conditions. For example, certain marine creatures have adapted to life at high pressures and salinity by using osmolytes to maintain cellular volume and buoyancy (1, 2). However, certain osmolytes, like urea, can degrade protein function and disrupt their structures at the high concentrations found in some animals and marine life (1-3), although elevated pressures (Ϸ70 MPa) could mitigate some of the deleterious effects of urea (4-6). Nevertheless, the answer to this paradox was the discovery of protective osmolytes such as betaine and trimethylamine N-oxide (TMAO) in certain elasmobranchs by Yancey et al. (1,[7][8] and later in other marine organisms and mammals (1,6,9). In marine animals, the TMAO concentration varies with habitat depth, presumably as a response to pressure (4-5). Furthermore, in organisms that concentrate urea as an osmolyte (7) and buoyancy factor (2), TMAO has been found in urea at ratios of 3:1 and 2:1 (7, 10).TMAO can restore enzyme function that has been lost because of the presence of urea (6, 10-12) by restoring the protein to its native structure (13-15). The mechanism of action of these protective osmolytes is not understood fully; both direct (16)(17)(18)(19) and indirect (13,(19)(20)(21) interactions have been proposed, and the mechanism may be molecule-specific (14). Our understanding of the mechanism of action of chemical denaturants, such as urea and guanidinium chloride, is in a similar state (19,20,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Consequently, we are pursuing molecular dynamics (MD) simulations of such compounds in an attempt to characterize these mechanisms at the molecular level.Here, we investigate the ability of TMAO to overcome the effect of urea on protein structure at the atomic level. Chymotrypsin inhibitor 2 (CI2) was chosen for this study because of the extensive amoun...

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“…Thus, this compound can exist in zwitterionic and positively charged forms, depending on the pH of the medium. Although TMAO-facilitated stabilization of proteins has been studied at length (8,(11)(12)(13)27), these studies were carried out in the pH range 6.0 -8.0 in which TMAO is almost neutral. To date, no studies have been carried out at pH values below the pK a of TMAO, where the osmolyte exists predominantly in the positively charged form.…”

mentioning

confidence: 99%

“…The effect of TMAO on protein stability and enzyme activity has been widely studied. This osmolyte has been shown in vitro to do the following: (i) increase the melting temperature as well as the unfolding free energy of proteins (7)(8)(9)(10)(11); (ii) offset the destabilizing effects of urea (8,10,11); (iii) restore the enzyme activity that is lost upon urea treatment (12,13); (iv) force the folding of unstructured proteins (4,(12)(13)(14)(15); (v) favor the protein self-association and polymerization of microtubules (16 -18); (vi) correct temperature-sensitive folding defects (19); and (vii) interfere with the formation of scrape prion protein (20). TMAO has been shown in vivo to counteract the damaging effects of salts (21), hydrostatic pressure (22,23), and urea (24,25) on proteins.…”

mentioning

confidence: 99%

Counteracting Osmolyte Trimethylamine N-Oxide Destabilizes Proteins at pH below Its pK

Singh

Haque

Ahmad

2005

Journal of Biological Chemistry

110

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Earlier studies have reported that trimethylamine Noxide (TMAO), a naturally occurring osmolyte, is a universal stabilizer of proteins because it folds unstructured proteins and counteracts the deleterious effects of urea and salts on the structure and function of proteins. This conclusion has been reached from the studies of the effect of TMAO on proteins in the pH range 6.0 -8.0. In this pH range TMAO is almost neutral (zwitterionic form), for it has a pK a of 4.66 ؎ 0.10. We have asked the question of whether the effect of TMAO on protein stability is pH-dependent. To answer this question we have carried out thermal denaturation studies of lysozyme, ribonuclease-A, and apo-␣-lactalbumin in the presence of various TMAO concentrations at different pH values above and below the pK a of TMAO. The main conclusion of this study is that near room temperature TMAO destabilizes proteins at pH values below its pK a , whereas it stabilizes proteins at pH values above its pK a . This conclusion was reached by determining the T m (midpoint of denaturation), ⌬H m (denaturational enthalpy change at T m ), ⌬C p (constant pressure heat capacity change), and ⌬G D°( denaturational Gibbs energy change at 25°C) of proteins in the presence of different TMAO concentrations. Other conclusions of this study are that T m and ⌬G D°d epend on TMAO concentration at each pH value and that ⌬H m and ⌬C p are not significantly changed in the presence of TMAO.Many organisms are known to accumulate low molecular weight organic molecules (osmolytes) in their tissues in response to harsh environmental stresses. These osmolytes are generally categorized into three groups, namely amino acids and their derivatives, polyhydric alcohols, and methylamines (1). Molecules of the first two groups are "compatible osmolytes," which means that cells accumulate these osmolytes to high concentrations without significantly perturbing protein functions under physiological conditions (1-4). Molecules of the third group, which reverse the perturbations caused by urea, are known as "counteracting osmolytes" (2, 5). One such counteracting osmolyte is trimethylamine N-oxide (TMAO), 1 which is present in high concentrations in coelacanth (sharks) and marine elasmobranchs (rays) (6). The effect of TMAO on protein stability and enzyme activity has been widely studied. This osmolyte has been shown in vitro to do the following: (i) increase the melting temperature as well as the unfolding free energy of proteins (7-11); (ii) offset the destabilizing effects of urea (8, 10, 11); (iii) restore the enzyme activity that is lost upon urea treatment (12, 13); (iv) force the folding of unstructured proteins (4, 12-15); (v) favor the protein self-association and polymerization of microtubules (16 -18); (vi) correct temperature-sensitive folding defects (19); and (vii) interfere with the formation of scrape prion protein (20). TMAO has been shown in vivo to counteract the damaging effects of salts (21), hydrostatic pressure (22, 23), and urea (24, 25) on proteins.TMAO is a compou...

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Time-Dependent Effects of Trimethylamine-N-Oxide/Urea on Lactate Dehydrogenase Activity: An Unexplored Dimension of the Adaptation Paradigm

Cited by 26 publications

References 22 publications

Natural Osmolyte Trimethylamine N-Oxide Corrects Assembly Defects of Mutant Branched-chain α-Ketoacid Decarboxylase in Maple Syrup Urine Disease

Natural Osmolyte Trimethylamine N-Oxide Corrects Assembly Defects of Mutant Branched-chain α-Ketoacid Decarboxylase in Maple Syrup Urine Disease

Counteraction of urea-induced protein denaturation by trimethylamine N -oxide: A chemical chaperone at atomic resolution

Counteracting Osmolyte Trimethylamine N-Oxide Destabilizes Proteins at pH below Its pK

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