Effects of osmolytes on hexokinase kinetics combined with macromolecular crowding: test of the osmolyte compatibility hypothesis towards crowded systems

We investigated the effect of compatible and non-compatible osmolytes in combination with macromolecular crowding on the kinetics of yeast hexokinase. This was motivated by the fact that almost all studies concerning the osmolyte effects on enzyme activity have been performed in diluted buffer systems, which are far from the physiological conditions within cells, where the cytosol contains several hundred mg protein ml(-1). Four organic (glycerol, betaine, TMAO and urea) and one inorganic (NaCl) osmolyte were tested. It was concluded that the effect of compatible osmolytes (glycerol, betaine and TMAO) on V(max) and K(M) was practically equivalent in pure buffer and in 200-250 mg BSA ml(-1) supporting the view that these small organic osmolytes do minimal perturbance on enzyme function in physiological solutions. The effect of urea on enzyme kinetics was not independent of protein concentration, since the presence of 250 mg BSA ml(-1) partly compensated the perturbing effect of urea. Even though the organic osmolytes glycerol, betaine and TMAO are generally considered compatible with enzyme function, especially glycerol did have a significant effect on hexokinase kinetics, decreasing both k(cat), K(M) and k(cat)/K(M). The osmolytes decreased k(cat)/K(M) in the order: NaCl>Urea>TMAO/glycerol>betaine. For the organic osmolytes this order correlates with the degree of exclusion from protein-water interfaces. Thus, the stronger the exclusion the weaker the perturbing effects on k(cat)/K(M).     

Compatibility of osmolytes with Gibbs energy of stabilization of proteins

This study led to the conclusion that naturally occurring osmolytes which are known to protect proteins against denaturing stresses, do not perturb the Gibbs energy of stabilization of proteins at 25 degrees C (DeltaG(D) degrees ) which has been shown to control the in vivo rate of degradative protein turnover (Pace et al., Acta Biol. Med. Germ 40 (1981) 1385-1392). This conclusion has been reached from our studies of heat-induced denaturation of lysozyme, ribonuclease A, cytochrome c and myoglobin in the presence of different concentrations of osmolytes, namely, glycine, proline, sarcosine and glycine-betaine. At a fixed concentration of osmolyte a heat-induced denaturation curve measured by following changes in the molar absorption coefficient of the protein, was analyzed for T(m), the midpoint of the denaturation and DeltaH(m), the enthalpy change of denaturation at T(m). Values of DeltaG(D) degrees were determined with Gibbs-Helmoltz equation using known values of T(m), DeltaH(m) and DeltaC(p), the constant-pressure heat capacity change. It has been observed that T(m) increases with the osmolyte concentration, whereas DeltaG(D) degrees remains unaffected in the presence of the osmolyte. This observation on DeltaG(D) degrees in the presence of osmolytes has been considered in the physiological context.     

Testing polyols' compatibility with Gibbs energy of stabilization of proteins under conditions in which they behave as compatible osmolytes

It is generally believed that compatible osmolytes stabilize proteins by shifting the denaturation equilibrium, native state <--> denatured state toward the left. We show here that if osmolytes are compatible with the functional activity of the protein at a given pH and temperature, they should not significantly perturb this denaturation equilibrium under the same experimental conditions. This conclusion was reached from the measurements of the activity parameters (K(m) and k(cat)) and guanidinium chloride-induced denaturations of lysozyme and ribonuclease-A in the presence of five polyols (sorbitol, glycerol, mannitol, xylitol and adonitol) at pH 7.0 and 25 degrees C.     

The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos

Betaine and proline protect preimplantation mouse embryos against increased osmolarity and decreased cell volume, implying that they may function as organic osmolytes. However, the transport system(s) that mediates their accumulation in fertilized eggs and early embryos was unknown, and previously identified mammalian organic osmolyte transporters could not account for their transport. Here, we report that there is a single saturable transport component shared by betaine and proline in 1-cell mouse embryos. A series of inhibitors had nearly identical effects on both betaine and proline transport by this system. In addition, K(i) values for reciprocal inhibition of betaine and proline transport were approximately 100-300 microM, similar to K(m) values ( approximately 200-300 microM) for their transport, and both had similar maximal transport rates (V(max)). The K(i) values for inhibition of betaine and proline transport by dimethylglycine were similar ( approximately 2 mM), further supporting transport of both substrates by a single transport system. Finally, betaine and proline transport each required Na(+)- and Cl(-). These data were consistent with a single, Na(+)- and Cl(-)-requiring, betaine/proline transport system in 1-cell mouse embryos. While betaine was only transported by a single saturable system, we found an additional, less conspicuous proline transport route that was betaine-insensitive, Na(+)-sensitive, and inhibited by alanine, leucine, cysteine, and methionine. Furthermore, we showed that betaine, like proline, is present in the mouse oviduct and thus could serve as a physiological substrate. Finally, accumulation of both betaine and proline increased with increasing osmolarity, consistent with a possible role as organic osmolytes in early embryos.     

Catalytic properties of the PepQ prolidase from Escherichia coli

The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with a proline residue at the C-terminus. The pepQ gene has been cloned, overexpressed, and the enzyme purified to homogeneity. The k(cat) and k(cat)/K(m) values for the hydrolysis of Met-Pro are 109 s(-1) and 8.4 x 10(5)M(-1)s(-1), respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group were assessed as substrates for PepQ. The S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a k(cat) of 36 min(-1) and a k(cat)/K(m) of 710 M(-1)s(-1). The corresponding R(P)-enantiomer was hydrolyzed more slowly with a k(cat) of 0.4 min(-1) and a k(cat)/K(m) of 11 M(-1)s(-1). The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX.     

Seasonal changes in the levels of compatible osmolytes in three halophytic species of inland saline vegetation in Hungary

Seasonal changes in the leaf concentration of compatible osmolytes were investigated in three halophytic species (Lepidium crassifolium, Camphorosma annua and Limonium gmelini subsp. hungaricum) native to a salty-sodic grassland. The investigated species were shown to accumulate both carbohydrate- and amino acid-derived osmolytes. The leaf tissues of C. annua (Chenopodiaceae) preferentially stored glycine betaine and pinitol, while in L. gmelini (Plumbaginaceae) beta-alanine betaine, choline-O-sulphate, and pinitol were accumulated. In the leaves of L. crassifolium (Brassicaceae) a very high amount of proline, associated with a high level of soluble carbohydrates was found. Not only the biochemical nature of the osmolyte, but also the seasonal pattern of osmolyte accumulation showed significant species-specific fluctuations. In addition, the cellular levels of the observed osmolytes changed with the growth period and according to the environmental parameters. The highest concentrations of osmolytes were found in March, when low temperatures, hypoxic conditions and high salt concentrations were the main constraints to plant growth. The high structural diversity of osmolytes combined with their multifunctionality and the seasonal flexibility of the metabolism in plants facing multiple stresses is discussed.     

Easy preparation of a large-size random gene mutagenesis library in Escherichia coli

Osmolytes are accumulated intracellularly to offset the effects of osmotic stress and protect cellular proteins against denaturation. Because different taxa accumulate different osmolytes, they can also be used as "dietary biomarkers" to study foraging. Potential osmolyte biomarkers include glycine betaine, trimethylamine N-oxide (TMAO), homarine, dimethylsulfoniopropionate (DMSP), and the osmolyte analog arsenobetaine (AsB). We present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of these osmolytes in serum or plasma. Varying concentrations of osmolytes were added to serum and samples and extracted in 90% acetonitrile and 10% methanol containing 10 μM deuterated internal standards (D(9)-glycine betaine, D(9)-trimethylamine-N-oxide, (13)C(2)-arsenobetaine, D(6)-DMSP, and D(4)-homarine). Analytes were separated on a normal-phase modified silica column and detected using isotope dilution tandem mass spectrometry in multiple reaction monitoring (MRM) mode. The assay was linear for all six compounds (r(2) values=0.983-0.996). Recoveries were greater than 85%, and precision for within-batch coefficients of variation (CVs) were less than 8.2% and between-batch CVs were less than 6.1%. Limits of detection ranged from 0.02 to 0.12 μmol/L. LC-MS/MS is a simple method with high throughput for measuring low levels of osmolytes that are often present in biological samples.     

The osmophobic effect: natural selection of a thermodynamic force in protein folding

Intracellular organic osmolytes are present in certain organisms adapted to harsh environments and these osmolytes protect intracellular macromolecules against the denaturing environmental stress. In natural selection of organic osmolytes as protein stabilizers, it appears that the osmolyte property selected for is the unfavorable interaction between the osmolyte and the peptide backbone, a solvophobic thermodynamic force that we call the osmophobic effect. Because the peptide backbone is highly exposed to osmolyte in the denatured state, the osmophobic effect preferentially raises the free energy of the denatured state, shifting the equilibrium in favor of the native state. By focusing the solvophobic force on the denatured state, the native state is left free to function relatively unfettered by the presence of osmolyte. The osmophobic effect is a newly uncovered thermodynamic force in nature that complements the well-recognized hydrophobic interactions, hydrogen bonding, electrostatic and dispersion forces that drive protein folding. In organisms whose survival depends on the intracellular presence of osmolytes that can counteract denaturing stresses, the osmophobic effect is as fundamental to protein folding as these well-recognized forces.     

Role of arginine 59 in the gamma-class carbonic anhydrases.

The functional role of the highly conserved active site Arg 59 in the prototype of the gamma-class carbonic anhydrase Cam (carbonic anhydrase from Methanosarcina thermophila) was investigated. Variants (R59A, -C, -E, -H, -K, -M, and -Q) were prepared by site-directed mutagenesis and characterized by size exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, and stopped-flow kinetic analyses. CD spectra indicated similar secondary structures for the wild type and the R59A and -K variants, independent of nondenaturing concentrations of guanidine hydrochloride (GdnHCl). SEC indicated that all variants purified as homotrimers like the wild type. SEC also revealed that the R59A and -K variants unfolded at > or = 1.5 M GdnHCl, compared to 3.0 M GdnHCl for the wild type. These results indicate that Arg 59 contributes to the thermodynamic stability of the Cam trimer. The R59K variant had k(cat) and k(cat)/K(m) values that were 8 and 5% of the wild-type values, respectively, while all other variants had k(cat) and k(cat)/K(m) values 10-100-fold lower than those of the wild type. The R59A, -C, -E, -M, and -Q variants exhibited 4-63-fold increases in k(cat) and 9-120-fold increases in k(cat)/K(m) upon addition of 100 mM GdnHCl, with the largest increases observed for the R59A variant, which was comparable to the R59K variant. The kinetic results indicate that a positive charge at position 59 is essential for the CO(2) hydration step of the overall catalytic mechanism.     

pH dependence and solvent deuterium oxide kinetic isotope effects on Bacillus cereus beta-lactamase I catalyzed reactions

The solvent kinetic isotope effects (SKIE's) on k(cat) (D(V)) and on k(cat/Km[D(V/K)] were determined for the Bacillus cereus beta-lactamase I catalyzed hydrolysis of five substrates that have values of k(cat)/K(m) varying over the range (0.014-46.3) X 10(6)M(-1) s(-1) and of k(cat) between 0.5 and 2019 s(-1). The variation of D(V/K) was only from 1.06 to 1.25 among these compounds and that in D(V) was from 1.50 to 2.16. These results require that Dk(1), the SKIE on the enzyme-substrate association rate constant, and D(k-1/k2), that on the partition ratio of the ES complex, both be near 1. The larger SKIE observed on D(V) requires that an exchangeable proton be in flight for either or both the acylation and the deacylation reaction. The pH dependence of the values k(cat)/K(m) for three substrates shows identical pK(a)s of 5.5. and 8.4. This identity combined with the fact that only one of these three substrates is kinetically "sticky" proves that the substrates can combine productively with only one protonic form of the enzyme. There is considerable substrate variation in the pK(a) values of k(cat) observed vs. pH profiles; the inflection points for all substrates studied are at pH values more extreme than are observed in the pH profiles for k(cat)/K(m).     

Nonfixed relationship of the Michaelis constant and maximum velocity with their corresponding rate constants

The Michaelis constant (K(m)) and V(mas) (E0k(cat)) values for two mutant sets of enzymes were studied from the viewpoint of their definition in a rapid equilibrium reaction model and in a steady state reaction model. The "AMP set enzyme" had a mutation at the AMP-binding site (Y95F, V67I, and V67I/L76V), and the "ATP set enzyme" had a mutation at a possible ATP-binding region (Y32F, Y34F, and Y32A/Y34A). Reaction rate constants obtained using steady state model analysis explained discrepancies found by the rapid equilibrium model analysis. (i) The unchanged number of bound AMPs for Y95F and the wild type despite the markedly increased K(m) values for AMP of the AMP set of enzymes was explained by alteration of the rate constants of the AMP step (k(+2), k(-2)) to retain the ratio k(+2)/k(-2). (ii) A 100 times weakened selectivity of ATP for Y34F in contrast to no marked changes in K(m) values for both ATP and AMP for the ATP set of enzymes was explained by the alteration of the rate constants of the ATP steps. A similar alteration of the K(m) and k(cat) values of these enzymes resulted from distinctive alterations of their rate constants. The pattern of alteration was highly suggestive. The most interesting finding was that the rate constants that decided the K(m) and k(cat) values were replaced by the mutation, and the simple relationships between K(m), k(cat), and the rate constants of K(m)1 = k(+1)/k(-1) and k(cat) = k(f) were not valid. The nature of the K(m) and k(cat) alterations was discussed.     

Dissecting the roles of osmolyte accumulation during stress

Many plants accumulate organic osmolytes in response to the imposition of environmental stresses that cause cellular dehydration. Although an adaptive role for these compounds in mediating osmotic adjustment and protecting subcellular structure has become a central dogma in stress physiology, the evidence in favour of this hypothesis is largely correlative. Transgenic plants engineered to accumulate proline, mannitol, fructans, trehalose, glycine betaine or ononitol exhibit marginal improvements in salt and/or drought tolerance. While these studies do not dismiss causative relationships between osmolyte levels and stress tolerance, the absolute osmolyte concentrations in these plants are unlikely to mediate osmotic adjustment. Metabolic benefits of osmolyte accumulation may augment the classically accepted roles of these compounds. In re-assessing the functional significance of compatible solute accumulation, it is suggested that proline and glycine betaine synthesis may buffer cellular redox potential. Disturbances in hexose sensing in transgenic plants engineered to produce trehalose, fructans or mannitol may be an important contributory factor to the stress-tolerant phenotypes observed. Associated effects on photoassimilate allocation between root and shoot tissues may also be involved. Whether or not osmolyte transport between subcellular compartments or different organs represents a bottleneck that limits stress tolerance at the whole-plant level is presently unclear. None the less, if osmolyte metabolism impinges on hexose or redox signalling, then it may be important in long-range signal transmission throughout the plant.     

Molecular mechanism for osmolyte protection of creatine kinase against guanidine denaturation.

The effects of osmolytes, including dimethysulfoxide, sucrose, glycine and proline, on the unfolding and inactivation of guanidine-denatured creatine kinase were studied by observing the fluorescence emission spectra, the CD spectra and the inactivation of enzymatic activity. The results showed that low concentrations of dimethysulfoxide (< 40%), glycine (< 1.5 m), proline (< 2.5 m) and sucrose (< 1.2 m) reduced the inactivation and unfolding rate constants of creatine kinase, increased the change in transition free energy of inactivation and unfolding (Delta Delta G(u)) and stabilized its active conformation relative to the partially unfolded state with no osmolytes. In the presence of various osmolytes, the inactivation and unfolding dynamics of creatine kinase were related to the protein concentrations. These osmolytes protected creatine kinase against guanidine denaturation in a concentration-dependent manner. The ability of the osmolytes to protect creatine kinase against guanidine denaturation decreased in order from sucrose to glycine to proline. Dimethysulfoxide was considered separately. This study also suggests that osmolytes are not only energy substrates for metabolism and organic components in vivo, but also have an important physiological function for maintaining adequate rates of enzymatic catalysis and for stabilizing the protein secondary and tertiary conformations.     

Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry

The hydrolysis of N-acetyl-L-methionine, N-acetylglycine, N-acetyl-L-phenylalanine, and N-acetyl-L-alanine at 298.35K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant (K(m)) and the catalytic constant (k(cat)) were determined from the progress of the reaction curve employing the integrated form of the Michaelis-Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k(cat) were up to a factor of 2.3 larger than those values determined from reciprocal initial velocity-initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K(m) were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600+/-31M(-1), determined by isothermal titration calorimetry, was used in the progress curve analysis, values for K(m) and k(cat) were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, Delta(r)H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from -4.69+/-0.09kJmol(-1) for N-acetyl-L-phenylalanine to -1.87+/-0.23kJmol(-1) for N-acetyl-L-methionine.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.     

Osmolytes protect mitochondrial F(0)F(1)-ATPase complex against pressure inactivation

We have previously reported that carbohydrates and polyols protect different enzymes against thermal inactivation and deleterious effects promoted by guanidinium chloride and urea. Here, we show that these osmolytes (carbohydrates, polyols and methylamines) protect mitochondrial F(0)F(1)-ATPase against pressure inactivation. Pressure stability of mitochondrial F(0)F(1)-ATPase complex by osmolytes was studied using preparations of membrane-bound submitochondrial particles depleted or containing inhibitor protein (IP). Hydrostatic pressure in the range from 0.5 to 2.0 kbar causes inactivation of submitochondrial particles depleted of IP (AS particles). However, the osmolytes prevent pressure inactivation of the complex in a dose-dependent manner, remaining up to 80% of hydrolytic activity at the highest osmolyte concentration. Submitochondrial particles containing IP (MgATP-SMP) exhibit low ATPase activity and dissociation of IP increases the hydrolytic activity of the enzyme. MgATP-SMP subjected to pressure (2.2 kbar, for 1 h) and then preincubated at 42 degrees C to undergo activation did not have an increase in activity. However, particles pressurized in the presence of 1.5 M of sucrose or 3.0 M of glucose were protected and after preincubation at 42 degrees C, showed an activation very similarly to those kept at 1 bar. In accordance with the preferential hydration theory, we believe that osmolytes reduce to a minimum the surface of the macromolecule to be hydrated and oppose pressure-induced alterations of the native fold that are driven by hydration forces.     

Diffusion-limited component of reactions catalyzed by Bacillus cereus beta-lactamase I

The Bacillus cereus beta-lactamase I catalyzes the hydrolysis of a wide variety of penicillins and cephalosporins with values of k(cat)/K(m) varying over several orders of magnitude. The values of this parameter for the most reactive of these compounds, benzylpenicillin, I, and furylacryloyl-penicillin, II (k(cat)/K(m) = 2.43 x 10(7) M(-1) s(-1) and 2.35 x 10(7) M(-1) s(-1), respectively, at pH 7.0 in potassium phosphate buffer containing 0.17 M KCl, I(c) = 0.63, 25 degrees C) are decreased markedly by increasing viscosity in sucrose- or glycerol-containing buffers. The relative sensitivities to viscosity of k(cat)/K(m) values for I and for cephaloridine, III, were found to be virtually unchanged at pH 3.8 from those observed at pH 7.0. The differential effects of viscosity on the reactive vs. the sluggish [e.g., cephalothin (IV), k(cat)/K(m) = 1 x 10(4) M(-1) s(-1)] substrates support the contention that the rates of reaction of the former with the enzyme are in part diffusion controlled. Quantitative analysis gives values for the association rate constants, k(1), of 7.6 x 10(7) M(-1) s(-1), 4 x 10(7) M(-1) s(-1), and 1.1 x 10(7) M(-1) s(-1) for I, II, and III, respectively. As both reactive and sluggish substrates associate with the active site of the enzyme with relatively similar rate constants, the variation in k(cat)/K(m) values is primarily due to the variation in the partition ratios k(-1)/k(2), for the ES complex, which are 2.3, 0.77, and 30 for I, II, and III, respectively. The preceding analysis is based on direct application of the Stokes-Einstein diffusion law to enzyme kinetics. The range of applicability of this law to the diffusion of substrate size molecules and the mechanics of diffusion of ionic species through viscous solutions of sucrose vs. polymers are explored.     

Osmolyte-induced protein folding free energy changes

Upon addition of protecting osmolyte to an aqueous solution of an intrinsically unstructured protein, spectral observables are often seen to change in a sigmoid fashion as a function of increasing osmolyte concentration. Commonly, such data are analyzed using the linear extrapolation model (LEM), a method that defines a scale from 0%-100% folded species at each osmolyte concentration by means of extending pre- and post-folding baselines into the transition region. Defining the 0%-100% folding scale correctly for each osmolyte is an important part of the analysis, leading to evaluation of the fraction of folded protein existing in the absence of osmolytes. In this study, we used reduced and carboxyamidated RNase T1 (RCAM-T1) as an intrinsically unstructured protein, and determined the thermodynamic stability of RCAM-T1 induced by naturally occurring osmolytes. Because the folded fraction of the protein population determined by experiments of thermal and urea-induced denaturation is nonzero in the absence of osmolytes at 15 degrees C, the commonly used LEM can lead to false values of DeltaG[stackD-->N0] for protein folding due to the arbitrary assumption that the protein is 100% unfolded in the presence of buffer alone. To correct this problem, titration of the protein solution with urea and extrapolating back to zero urea concentration gives the spectral value for 100% denatured protein. With fluorescence as the observable we redefine F/F0 to F/F0extrap = 1.0 and require that the denatured-state baseline have this value as its intercept. By so doing, the 0%-100% scale-corrected DeltaG[D-->N0] values of RCAM-T1 folding in the presence of various osmolytes are then found to be identical, with small error, demonstrating that DeltaG[D-->N0] is independent of the osmolytes used. Such a finding is an important step in validating this quantity derived from the LEM as having the properties expected of an authentic thermodynamic parameter. The rank order of osmolyte efficacies in stabilizing RCAM-T1 is sarcosine > sucrose > sorbitol > proline > betaine > glycerol.     

The pyruvate kinase model system, a cautionary tale for the use of osmolyte perturbations to support conformational equilibria in allostery

In the study of rabbit muscle pyruvate kinase (M₁‐PYK), proline has previously been used as an osmolyte in an attempt to determine a role for preexisting conformational equilibria in allosteric regulation. In this context, osmolytes are small molecules assumed to have no direct interaction with the protein. In contrast to proline's proposed role as an osmolyte, the structure of M₁PYK‐Mn‐pyruvate‐proline complex reported herein demonstrates that proline binds specifically to the allosteric site of M₁‐PYK. Therefore, this amino acid is an allosteric effector rather than a benign osmolyte. Other compounds often used as osmolytes (polyethyleneglycol and glycerol) are also present in the structure, suggesting an interaction with the protein that would, in turn, prevent the usefulness of these compounds in the study of this and most likely other proteins. These findings highlight the need to verify that compounds used as osmolytes to perturb preexisting conformational equilibrium do not directly interact with the protein, a consideration not commonly addressed in the past.