Background: Covalent modification cycles are widely used as regulatory switches. Results: Mathematical analysis reveals, under very general assumptions, an unavoidable trade-off between switching efficiency and cell-to-cell coherence. Conclusion: Enzyme bifunctionality offers a way to circumvent this trade-off. Significance: This may explain the bifunctionality of PFK-2/FBPase-2 in controlling the switch between glycolysis and gluconeogenesis in the mammalian liver.Covalent modification provides a mechanism for modulating molecular state and regulating physiology. A cycle of competing enzymes that add and remove a single modification can act as a molecular switch between "on" and "off" and has been widely studied as a core motif in systems biology. Here, we exploit the recently developed "linear framework" for time scale separation to determine the general principles of such switches. These methods are not limited to Michaelis-Menten assumptions, and our conclusions hold for enzymes whose mechanisms may be arbitrarily complicated. We show that switching efficiency improves with increasing irreversibility of the enzymes and that the on/off transition occurs when the ratio of enzyme levels reaches a value that depends only on the rate constants. Fluctuations in enzyme levels, which habitually occur due to cellular heterogeneity, can cause flipping back and forth between on and off, leading to incoherent mosaic behavior in tissues, that worsens as switching becomes sharper. This trade-off can be circumvented if enzyme levels are correlated. In particular, if the competing catalytic domains are on the same protein but do not influence each other, the resulting bifunctional enzyme can switch sharply while remaining coherent. In the mammalian liver, the switch between glycolysis and gluconeogenesis is regulated by the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/ FBPase-2). We suggest that bifunctionality of PFK-2/FBPase-2 complements the metabolic zonation of the liver by ensuring coherent switching in response to insulin and glucagon.An enzyme-catalyzed modification cycle is illustrated in Fig. 1A. The forward enzyme, E, catalyzes the covalent addition of an M moiety (phosphoryl, methyl, acetyl, etc.), carried by the donor, D-M, to form the modified substrate, S 1 , from the unmodified substrate, S 0 . The reverse enzyme, F, catalyzes the removal of M, returning S 1 to S 0 . Background metabolic processes continually replenish D-M from D and M. The substrate, S, can be any molecule, protein or otherwise.Such cycles can function as biological switches, in which the proportion of S 1 at steady state can be varied from low ("off") to high ("on") by altering properties of the cycle, such as the enzyme levels (1, 2). They are regarded as core motifs in cellular information processing (3,4) and have been the subject of much analysis (5-9).Application of these results to specific biological examples has been hampered, however, by the universal assumption that the enzymes E and F follow the Mich...
The phase behavior of cholesteryl esters with triglyceride has been characterized by differential scanning calorimetry (DSC), light microscopy, and polarizing light microscopy (PLM). Temperature-dependent molecular motions determined by 13C NMR spectroscopy were correlated with thermotropic phase behavior. Two systems, cholesteryl oleate (CO) and a 3/1 w/w mixture of cholesteryl linoleate (CL) and CO, were examined in the presence of small amounts of triolein (TO). Both systems exhibited metastable cholesteric and smectic (or only smectic) phases. Increasing amounts of TO progressively lowered the liquid-crystalline phase transition temperatures and eventually abolished the cholesteric phase, but at differing amounts of TO for the two systems (between 4% and 5% with CL/CO and between 7% and 10% with CO). DSC and PLM showed a progressive broadening of the phase transitions as well as an overlapping of the temperature ranges of the cholesteric and smectic phases. At greater than or equal to 4% TO, a separate isotropic liquid phase coexisted with liquid-crystalline phases. 13C NMR spectroscopy was used to monitor the molecular motions of the cholesteryl ester steroid ring and acyl chain in liquid and liquid-crystalline phases. In the liquid phase, no significant changes in fatty acyl motions, as reflected in spin-lattice relaxation time (T1) and nuclear Overhauser enhancement (NOE) values, were found on addition of TO. The line width (v 1/2) of the steroid ring resonances increased markedly near (1-5 degrees C above) the isotropic liquid----liquid-crystal phase transition temperature (TLC). However, the C3/C6 v 1/2 ratio at 1 degree C above TLC was greater for mixtures exhibiting an isotropic----cholesteric transition than for mixtures exhibiting an isotropic----smectic transition. Rotational correlation times calculated for motions about the long molecular axis and the nonunique axis showed (i) that the ring motions became more anisotropic as TLC was approached and (ii) that the motions were more anisotropic at TLC + 1 degree C for systems exhibiting a cholesteric phase than for systems exhibiting only a smectic phase. 13C line widths in spectra of the cholesteryl ester liquid-crystalline phases suggested that TO perturbed the cholesteryl ester intermolecular interactions and increased the rates of cholesteryl ester molecular motions relative to neat esters.
Peaks corresponding to the C6 protons of the four histidine residues (positions 58, 111, 123, and 182) of chicken ovomucoid have been assigned in 1H NMR spectra (360 or 470 MHz) of the native single-chain protein and of fragments of the protein corresponding to its three homologous structural domains. Comparison of the 1H NMR pH titration behavior of these histidine residues and the deuterium exchange rates of their C6-H positions show the following: (1) The chemical shift properties of histidine residues 58, 123, and 182 differ despite the fact that the three residues are located in homologous positions in the three tandem domains. (2) The properties of three of the four histidine residues (58, 111, and 123) do not change appreciably when the domains in which they are located are isolated, indicating that their environments are similar in both the fragment and the native protein. (3) The properties of the fourth histidine (182) differ in the isolated domain and in the native protein. (4) The observed properties of the histidine residues stem primarily from intradomain interactions that remain constant in isolated domains rather than from interactions with neighboring domains; an interdomain interaction is required to explain the behavior of only histidine-182. (5) The chemical shift of histidine-111 is affected by the titration of the side chain of aspartate-98 with pHmid 2.6 in native ovomucoid but not in isolated second domain; the chemical shift of histidine-182 is perturbed by the titration of the carboxyl group of the C-terminal cysteine-186 with pHmid 2.4 in native ovomucoid and pHmid 2.6 in isolated third domain.
To examine the validity of the deuterium (D) incorporation technique for measurement of human cholesterol synthesis rates, D uptake from D 2 O into cholesterol was compared to sterol balance in 13 subjects each under three controlled diet settings. Subjects (age 62 ؎ 3.6 yr, body weight 74 ؎ 4.0 kg, BMI 27 ؎ 1.4) consumed weight maintenance diets enriched in either corn oil, beef tallow, or stick corn oil margarine over a 5-week period. During the final week of the study period, subjects were given 1.2 g/ D 2 O per kg body water. D enrichment was measured in plasma water and total cholesterol over 24 h. Also, during the final week, dietary intake and fecal elimination rates of cholesterol were assessed over one 6-day period to calculate sterol balance. There was no significant difference (t ؍ 0.858, P ؍ 0.397) between D incorporation into cholesterol (1,183 ؎ 92 mg/day) and sterol balance (1,316 ؎ 125 mg/ day). Among diets, net cholesterol biosynthesis measured by D incorporation agreed ( r ؍ 0.745, P ؍ 0.0001) with values derived from sterol balance. The degree of association between methods was not influenced by the wide range of fatty acid composition of the diet fat. These data demonstrate the utility of the simple, non-restrictive deuterium incorporation method as a reliable means of determining cholesterol biosynthesis in free-living humans.-Jones, P.
Articles you may be interested inTemperature-dependent shear viscosity coefficient of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX): A molecular dynamics simulation study High resolution l3C NMR spectra were obtained vs temperature in the isotropic liquid and liquid-crystalline phases of four cholesteryl esters with saturated acyl chains [( 6:0), (14:0), (16:0), and (18:0)] and a mixture rich in cholesteryl esters with unsaturated acyl chains [10% 16:0,45% 18:1,45% 18:2, w/w/w]. Except for peaks from carbons in the middle and/ or distal part of the fatty acyl chain, the narrow peaks seen for the isotropic liquid broadened beyond detection below the isotropic->cholesteric phase transition, indicating a marked decrease in the rates and/or amplitudes of motions of most portions of the cholesteryl ester molecule. Rotational correlation times for the long molecular axis and the short non unique axis ('Trz and 'T rx ' respectively), were calculated from the linewidth of the steroid ring methine C3 and C6 resonance for each ester system in the liquid phase. With decreasing temperature, 'T rx and'T rz showed little or no increase for the saturated esters but a significant differential increase for the mixture. Because of stronger intermolecular interactions of neat saturated esters, molecular ordering near the liquid-liquid-crystalline phase transition may occur in such a narrow temperature range that the large pretransitional anisotropies observed for unsaturated esters are not observed for the saturated esters. 7380 J. Chem. Phys. 85 (12). 15 December 1986
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