Fatty acid trafficking in the adipocyte.

The insolubility of fatty acids in cellular environments requires that specific trafficking mechanisms be developed to vectorally orient and deliver lipids for cellular needs. The roles of putative membrane bound fatty acid transporters and soluble carrier proteins are discussed in terms of mechanisms of fatty acid trafficking. The numerous roles for fatty acids as an energy source, as structural elements for membrane synthesis, as bioregulators and as prohormones with the potential to regulate gene expression, are discussed in terms of the necessity to regulate their intracellular location and concentration.

Biochemical and biophysical analysis of the intracellular lipid binding proteins of adipocytes.

Adipocytes express two lipid-binding proteins; the major one termed the adipocyte lipid-binding protein or aP2 (ALBP/aP2) and a minor one referred to as the keratinocyte lipid-binding protein (KLBP). In order to evaluate the potential physiological roles for these proteins, their biochemical and biophysical properties have been analyzed and compared. ALBP/aP2 and KLBP exhibit similar binding affinities for most long-chain fatty acids; however, ALBP/aP2 exhibits a two to three-fold increased affinity for myristic, palmitic, oleic and linoleic acids, the predominant fatty acids of adipocytes. As measured by guanidinium hydrochloride denaturation, the stability of ALBP/aP2 is nearly 3 kcal/mol greater than that of KLBP. While the pI of ALBP/aP2 was determined to be 9.0, that of KLBP is 6.5 suggesting differing net charges at physiological pH. Analysis of surface electrostatic properties of ALBP/aP2 and KLBP revealed similar charge polarity, although differences in the detailed charge distribution exist between the proteins. The distribution of hydrophobic patches was also different between the proteins,ALBP/aP2 has only scattered hydrophobic surfaces while KLBP has a large hydrophobic patch near the ligand portal into the binding cavity. In sum, these results point out that despite the striking similarity between ALBP/aP2 and KLBP in tertiary structure, significant differences in ligand binding and surface properties exist between the two proteins. Hence, while it is tempting to speculate that ALBP/aP2 and KLBP are metabolically interchangeable, careful analysis suggests that the two proteins are quite distinct and likely to play unique metabolic roles.

The magnitude of the allosteric conformational transition of aspartate transcarbamylase is altered by mutations.

Global conformational transitions are of central functional importance for many enzymes and binding proteins. It is not known, however, how much variability can exist in such structural-functional linkages. We have characterized the global magnitude of the T to R conformational transition of Escherichia coli aspartate transcarbamylase (ATCase) by measuring (1) hydration changes using osmotic stress and (2) hydrodynamic changes using high-precision analytical gel chromatography. We find that specific mutations can alter the structural magnitude of the enzyme's conformational transition without abolishing allostery, suggesting that some degree of plasticity exists in the conformational component of allostery.

Surface properties of adipocyte lipid-binding protein: Response to lipid binding, and comparison with homologous proteins.

Adipocyte lipid-binding protein (ALBP) is one of a family of intracellular lipid-binding proteins (iLBPs) that bind fatty acids, retinoids, and other hydrophobic ligands. The different members of this family exhibit a highly conserved three-dimensional structure; and where structures have been determined both with (holo) and without (apo) bound lipid, observed conformational changes are extremely small (Banaszak, et al., 1994, Adv. Prot. Chem. 45, 89; Bernlohr, et al., 1997, Annu. Rev. Nutr. 17, 277). We have examined the electrostatic, hydrophobic, and water accessible surfaces of ALBP in the apo form and of holo forms with a variety of bound ligands. These calculations reveal a number of previously unrecognized changes between apo and holo ALBP, including: 1) an increase in the overall protein surface area when ligand binds, 2) expansion of the binding cavity when ligand is bound, 3) clustering of individual residue exposure increases in the area surrounding the proposed ligand entry portal, and 4) ligand-binding dependent variation in the topology of the electrostatic potential in the area surrounding the ligand entry portal. These focused analyses of the crystallographic structures thus reveal a number of subtle but consistent conformational and surface changes that might serve as markers for differential targeting of protein-lipid complexes within the cell. Most changes are consistent from ligand to ligand, however there are some ligand-specific changes. Comparable calculations with intestinal fatty-acid-binding protein and other vertebrate iLBPs show differences in the electrostatic topology, hydrophobic topology, and in localized changes in solvent exposure near the ligand entry portal. These results provide a basis toward understanding the functional and mechanistic differences among these highly structurally homologous proteins. Further, they suggest that iLBPs from different tissues exhibit one of two predominant end-state structural distributions of the ligand entry portal.

Solvent perturbation of the allosteric regulation of aspartate transcarbamylase.

Escherichia coli aspartate transcarbamylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis, the condensation of aspartate and carbamyl phosphate. ATCase is positively allosterically regulated by ATP and negatively regulated by CTP. We have used mild solvent perturbation to gain global molecular information about the mechanism of heterotropic allostery. The [NaCl], temperature, and osmotic pressure dependence of the enzymatic activity of ATCase has been examined in the presence and absence of allosteric effectors. The results indicate that: 1) Regulation of aspartate binding by CTP appears to involve a unique set of electrostatic interactions not involved in enzyme function in the presence of ATP or in the absence of effectors. 2) Aspartate binding is enthalpically driven in the presence and absence of allosteric effectors. 3) The apparent enthalpy and entropy of aspartate binding (delta H, delta S), and activation energy of catalysis (Ea) are substantially altered in the presence of CTP but not ATP. 4) The change in hydration of ATCase upon substrate binding is the same in the presence and absence of allosteric effectors. 5) The linkage between heterotropic and homotropic allostery is different for ATP and CTP.

Functionally linked hydration changes in Escherichia coli aspartate transcarbamylase and its catalytic subunit.

Aspartate transcarbamylase (ATCase) is a highly regulated, dodecameric enzyme that catalyzes the first committed step in pyrimidine biosynthesis. Upon ligation, ATCase undergoes a conformational transition from a low-activity T-state to a high-activity R-state. This transition involves major changes in the molecular architecture, including structural rearrangements of several intersubunit interfaces and a 12 A expansion of the molecule along its 3-fold axis. Solute-induced osmotic stress experiments report that approximately 208 solvent waters are taken up by ATCase as it binds substrate. Solvent-accessible surface area calculations conducted on the T and R conformers of ATCase agree very well with this result, predicting that approximately 189 waters are taken up during this conformational change. Both osmotic stress measurements and surface area calculations on the catalytic trimer of ATCase predict water release upon ligation of the trimer. Specific aspects of the application of osmotic stress to ATCase are also discussed, including solute size effects, and an assessment of potential alternative explanations for these results.

Local fluctuations and global unfolding of partially folded BPTI detected by NMR.

The protein [14-38]Abu is a chemically synthesized variant of bovine pancreatic trypsin inhibitor (BPTI) with the 14-38 disulfide bond intact and cysteines 5, 30, 51, and 55 replaced by alpha-amino-n-butyric acid (Abu). At 1-6 degrees C and pH 4.5-6.5, [14-38]Abu is partially folded with a native-like core [1]. Heteronuclear NMR spectra contain two, and in a few cases three or four, exchange cross peaks for each 15N-bound 1H, reporting the presence of two or more conformations that interconvert on a time scale of > or = milliseconds. Thermodynamic analysis of 15N-1H exchange peak volumes as a function of temperature in the range 1-35 degrees C indicates that partially folded [14-38]Abu undergoes local segmental motions as well as cooperative global unfolding. The relative abundance of more folded versus disordered conformations changes throughout the molecule, indicating that various regions of the partially folded protein are disordered to different extents prior to onset of thermal denaturation. This system is unique in providing a measure of the populations of interconverting partially folded conformations, as well as a microscopic view of cooperative folding of a fluctuating ensemble. Although global thermal denaturation is cooperative, significant deviation from simple two-state behavior is reflected in several parameters, including the difference in Tm for thermal unfolding measured by NMR versus circular dichroism.

Is substrate inhibition a consequence of allostery in aspartate transcarbamylase?

Aspartate transcarbamylase (ATCase) is a highly regulated, multisubunit enzyme that catalyzes the first regulated step in pyrimidine biosynthesis. Although ATCase exhibits strong substrate inhibition (the reduction of enzyme activity at high substrate concentrations), the mechanism of substrate inhibition has not been investigated. At the molecular level, substrate inhibition may result either from local events at the active site or from global or specific long-range allosteric effects. We have compared the results of fitting kinetic data to several models: (a) a semi-empirical steady-state kinetic model that includes cooperative substrate binding (described by a Hill coefficient) and partial uncompetitive substrate inhibition, (b) a nested allosteric model developed to analyze substrate inhibition of the ATPase activity of GroEL, an enzyme with a quaternary structure analogous to ATCase (O. Yifrach and A. Horovitz, Biochemistry, 34 (1995) 5303), and (c) purely concerted models, including a model originally proposed by Monod et al. (J. Monod, J. Wyman and J.P. Changeux, J. Mol. Biol., 12 (1965) 88). Model (a) is the first kinetic equation for ATCase that both fits the data and returns physically realistic values for all parameters, but it is a modified Hill equation and thus returns little or no molecular mechanistic information. The nested allosteric model (b), which assumes concerted cooperativity within each catalytic trimer of ATCase and sequential cooperativity between trimers, is unlikely to be the correct model for ATCase, since isolated catalytic trimers, which cannot exhibit the sequential cooperativity of the model, still exhibit substrate inhibition. Analysis of concerted models (c) shows that a two-state model is inadequate to account for substrate inhibition in ATCase. Further, although unique fits to a three-state model cannot be obtained, because the parameters are highly correlated, several sets of parameter values fit the data well and are in accord with other experimental results. These results indicate that substrate inhibition in ATCase may be the consequence of allostery, and that further experimental investigation is warranted.

Effects of assembly and mutations outside the active site on the functional pH dependence of Escherichia coli aspartate transcarbamylase.

Electrostatics are central to the function and regulation of Escherichia coli aspartate transcarbamylase, and modeling has suggested that long range electrostatic effects are likely to be important (Glackin, M. P., McCarthy, M. P., Mallikarachchi, D., Matthew, J. B., and Allewell, N. M. (1989) Proteins Struct. Funct. Genet. 5, 66-77; Oberoi, H., Trikha, J., Yuan, X., and Allewell, N. M. (1995) Proteins Struct. Funct. Genet., in press). To investigate this possibility from an experimental standpoint, we have examined the effects both of assembly and of removing ionizable and polar side chains outside the active site (Glu-50, Tyr-165, and Tyr-240) on the pH dependence of the kinetic parameters of aspartate transcarbamylase. The holoenzyme (c6r6) assembles from three regulatory dimers (r2) and two catalytically active trimers (c3). pH dependences of the enzyme kinetic parameters suggest that the mechanisms of productive binding of L-Asp to the binary complexes of the catalytic subunit (c3) and holoenzyme (c6r6) with carbamyl phosphate are different. In contrast, the Michaelis complex appears similar for both c3 and c6r6, except for pK shifts of approximately 1 pH unit. Results also indicate that the catalytic mechanism of the holoenzyme does not involve reverse protonation, as has recently been proposed for the catalytic trimer (Turnbull, J. L., Waldrop, G. L., and Schachman, H. K. (1992) Biochemistry 31, 6562-6569). The tyrosines at positions 165 and 240 are part of a cluster of interactions that links the catalytic subunits in the T state (the cluc4 interface) and which is disrupted in the T --> R transition. The effects of mutating the two Tyr residues are quite different: Y240F has higher than wild-type activity and affinity over the entire pH range, while Y165F has activity and affinity an order of magnitude lower than wild-type. Removal of the regulatory subunits from Y165F increases activity and affinity and restores the pH dependence of the wild-type catalytic subunit. Like Y165F, E50A has low activity and affinity over the entire pH range. Linkage analysis indicates that there is long range energetic coupling among the active site, the ear subunit interfaces, and residue Y165. The substantial quantitative difference between Y165F and Y240F, both of which are at the c1:c4 interface about 14-16 A from the closest active site, demonstrates specific path dependence, as opposed to general distance dependence, of interactions between this interface and the active site.

Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase.

We have examined the pathway and energetics of urea-induced dissociation and unfolding of the catalytic trimer (c3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N-(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl- and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active-site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for delta Gdissociation at 7.5 degrees C of about 7-10 kcal m-1 per interface. delta Gunfolding of dissociated chains when modeled as a 2-state process is estimated to be very small, on the order of -2 kcal m-1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1-state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl-, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand.

Single-site modifications of half-ligated hemoglobin reveal autonomous dimer cooperativity within a quaternary T tetramer.

The patterns of energetic response elicited by single-site hemoglobin mutations and chemical modifications have been determined in order to probe the dimer-dimer interface of the half-ligated tetramer (species [21]) that was previously shown to behave as allosterically distinct from both the unligated and fully ligated molecules. In this study the free energies of quaternary assembly (dimers to tetramers) were determined for a series of 24 tetrameric species in which one dimeric half-molecule is ligated (cyanomet hemes) while the adjacent alpha beta dimer is unligated and contains a single amino acid modification. Assembly energies have also been determined for tetramers bearing the same amino acid modifications but where the hemesites were completely vacant and additionally where they were fully occupied. A total of 72 molecular species were thus characterized. It was found that mutationally induced perturbations to the free energy of quaternary assembly were identical for the half-ligated tetramers and the unligated tetramers over the entire spatial distribution of altered sites, but exhibited a radically different pattern from that of the fully ligated molecules. These results indicate that the dimer-dimer interface of the half-ligated tetramer (species [21]) has the same quaternary structure as that of the unligated molecule, i.e., "quaternary T." This quaternary structure assignment of species [21] strongly supports the operation of a Symmetry Rule which translates changes in hemesite ligation into six T-->R quaternary switchpoints. Analysis of the observed Symmetry Rule behavior in relation to the measured distribution of cooperative free energies for the partially ligated species reveals significant cooperativity between alpha and beta subunits of the dimeric half-tetramer within quaternary T. The mutational results indicate that these interactions are not "paid for" by breaking or making noncovalent bonds at the dimer-dimer interface (alpha 1 beta 2). They arise from structural and energetic changes that are "internal" to the ligated dimer even though its association with the unligated dimer is required for the cooperativity to occur. Free energy of "tertiary constraint" is thus generated by the first binding step and is propagated to the second hemesite while the dimer-dimer interface alpha 1 beta 2 serves as a constraint. The "sequential" cooperativity that occurs within the half-molecule is thus preconditioned by the constraint of a quaternary T interface; release of this constraint by dissociation produces only noncooperative dimers.(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental resolution of cooperative free energies for the ten ligation species of cobalt(II)/iron(II)-CO hemoglobin.

Cooperative free energies have been determined for the 10 ligation species of human hemoglobin in the Co(II)/Fe(II)-CO system. In this system, subunits containing unligated cobaltous hemes coexist in the same tetramer with naturally occurring ferrous hemes that are ligated with carbon monoxide. Tetramers comprising the 10 structurally unique combinations of ligated and unligated subunits were characterized in terms of their dimer-tetramer assembly free energies. By use of the thermodynamic linkage between assembly and ligation, the experimentally resolved values were used to obtain the corresponding cooperative free energies (i.e., the differences between actual free energies of ligation and the summed contributions of intrinsic values). The results obtained are in general accord with previous findings on this same system (Imai et al., 1980). The present study extends this earlier work by resolving the cooperative properties of each configurational isomer of the doubly ligated tetramers. The 10 ligation species were found to distribute into 5 discrete cooperative free energy levels according to a combinatorial code which includes, as a special case, the code found previously with cyanomethemoglobin and manganese-substituted hemoglobin (Smith et al., 1987; Daugherty et al., 1991). This distribution exhibits additional characteristics found in the oxygenation of normal ferrous hemoglobin including the quaternary enhancement effect (Mills & Ackers, 1979a,b). These results, and those of the following paper (Doyle et al., 1991), strongly support the premise that a common set of qualitative rules governs the cooperative interactions in hemoglobin irrespective of

Linkage between cooperative oxygenation and subunit assembly of cobaltous human hemoglobin.

The thermodynamic linkage between cooperative oxygenation and dimer-tetramer subunit assembly has been determined for cobaltous human hemoglobin in which iron(II) protoporphyrin IX is replaced by cobalt(II) protoporphyrin IX. The equilibrium parameters of the linkage system were determined by global nonlinear least-squares regression of oxygenation isotherms measured over a range of hemoglobin concentrations together with the deoxygenated dimer-tetramer assembly free energy determined independently from forward and reverse reaction rates. The total cooperative free energy of tetrameric cobalt hemoglobin (over all four binding steps) is found to be 1.84 (+/- 0.13) kcal, compared with the native ferrous hemoglobin value of 6.30 (+/- 0.14) kcal. Detailed investigation of stepwise cooperativity effects shows the following: (1) The largest change occurs at the first ligation step and is determined on model-independent grounds by knowledge of the intermediate subunit assembly free energies. (2) Cooperativity in the shape of the tetrameric isotherm occurs mainly during the middle two steps and is concomitant with the release of quaternary constraints. (3) Although evaluation of the pure tetrameric isotherm portrays identical binding affinity between the last two steps, this apparent noncooperativity is the result of a "hidden" oxygen affinity enhancement at the last step of 0.48 (+/- 0.12) kcal. This quaternary enhancement energy is revealed by the difference in subunit assembly free energies of the triply and fully ligated species and is manifested visually by the oxygenation isotherms at high versus low hemoglobin concentration. (4) Cobaltous hemoglobin dimers exhibit apparent anticooperativity of 0.49 (+/- 0.16) kcal (presumed to arise from heterogeneity of subunit affinities).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

The 10 ligation species of human cyanomethemoglobin were previously found to distribute into three discrete cooperative free energy levels according to a combinatorial code (i.e., dependent on both the number and configuration of ligated subunits). Analysis of this distribution showed that the hemoglobin tetramer occupies a third allosteric state in addition to those of the unligated (T) and fully ligated (R) species. To determine the nature of the intermediate allosteric state, we have studied the effects of pH, temperature, and single-site mutations on its free energy of quaternary assembly, in parallel with corresponding data on the deoxy (T) and fully ligated (R) species. Results indicate that the intermediate allosteric tetramer has the deoxy (T) quaternary structure. This finding, together with the resolved energetic distribution of the 10 microstates reveals a symmetry rule for quaternary switching--i.e., switching from T to R occurs whenever a binding step creates a tetramer with one or more ligated subunits on each side of the alpha 1 beta 2 intersubunit contact. These studies also reveal significant cooperativity within each alpha 1 beta 1 dimer of the T-state tetramer. The ligand-induced tertiary free energy alters binding affinity within the T structure by 170-fold prior to quaternary switching.

Direct and indirect pathways of functional coupling in human hemoglobin are revealed by quantitative low-temperature isoelectric focusing of mutant hybrids.

Functional energetic coupling within human hemoglobin has been explored by using quantitative analysis of asymmetric mutant hybrid equilibria. Previous work showed that the free energy of cooperativity is largely attributable to alterations in free energy that accompany changing interactions at the interface between alpha 1 beta 1 and alpha 2 beta 2 dimers [Pettigrew et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1849]. However, the issue of how cooperativity-linked sites in the molecule are energetically coupled in manifesting cooperative ligation is still not well delineated. In this paper we address the questions of what types of functional coupling pathways are operational in hemoglobin, what some of their characteristics are, and how they are related to one another. By constructing asymmetric mutant hybrid hemoglobins, we can assay how two structurally identical, symmetrically equivalent sites are energetically coupled in manifesting subunit assembly and/or cooperative ligation. Asymmetric hybrid hemoglobins, i.e., those containing a single modified site, cannot be isolated and must be studied in equilibrium with their symmetric parent molecules. In order to study these asymmetric hybrid equilibria, we have developed new theory and quantitation techniques to augment the low-temperature quenching and isoelectric focusing procedures of Perrella et al. [(1978) Anal. Biochem. 88, 212]. Studies of these mutant hybrid hemoglobins have provided evidence for three distinct types of energetic coupling within the hemoglobin tetramer. All alpha 1 beta 2 interface sites examined are involved in cooperativity-linked indirect coupling. Within the context of this indirect "pathway" there exist two different types of direct long-range coupling. One of these classes of direct long-range pathways is linked to cooperative ligand binding while the other class is not.

Interaction of bovine mitochondrial ribosomes with Escherichia coli initiation factor 3 (IF3).

Mammalian mitochondrial ribosomes are distinguished from their bacterial and eukaryotic-cytoplasmic counterparts, as well as from mitochondrial ribosomes of lower eukaryotes, by their physical and chemical properties and their high protein content. However, they do share more functional homologies with bacterial ribosomes than with cytoplasmic ribosomes. To search for possible homologies between mammalian mitochondrial ribosomes and bacterial ribosomes at the level of initiation factor binding sites, we studied the interaction of Escherichia coli initiation factor 3 (IF3) with bovine mitochondrial ribosomes. Bacterial IF3 was found to bind to the small subunit of bovine mitochondrial ribosomes with an affinity of the same order of magnitude as that for bacterial ribosomes, suggesting that most of the functional groups contributing to the IF3 binding site in bacterial ribosomes are conserved in mitochondrial ribosomes. Increasing ionic strength affects binding to both ribosomes similarly and suggests a large electrostatic contribution to the reaction. Furthermore, bacterial IF3 inhibits the Mg2+-dependent association of mitochondrial ribosomal subunits, suggesting that the bacterial IF3 binds to mitochondrial small subunits in a functional way.