Thermodynamic nonideality as a probe of allosteric mechanisms: preexistence of the isomerization equilibrium for rabbit muscle pyruvate kinase

Sedimentation velocity studies in the presence and absence of an inert space-filling solute, sucrose, have been used to establish preexistence of the isomerization equilibrium responsible for the allosteric behavior of rabbit muscle pyruvate kinase. Whereas the inclusion of phenylalanine (5 mM) with enzyme gives rise to a decrease of 0.3 S in the sedimentation coefficient of pyruvate kinase, the corresponding effect of phosphoenolpyruvate is to increase the sedimentation coefficient by 0.03 S. Consideration of these findings to signify the existence of an isomeric equilibrium between compact and expanded forms of the enzyme is substantiated by the finding that inclusion of sucrose (0.1 M) also brings about the change in sedimentation coefficient effected by phosphoenolpyruvate. By demonstrating that rabbit muscle pyruvate kinase undergoes isomerization in the absence of substrate, this study removes any necessity to consider the existence of an isomerization equilibrium that is substrate-induced; and thereby provides experimental support for adoption of the Monod model of allostery to interpret enzyme kinetic data for pyruvate kinase [R. W. Oberfelder, B. G. Barisas, and J. C. Lee (1984) Biochemistry 23, 3822-3826].     

Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.     

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli.

The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism.     

Self-association of rabbit muscle phosphofructokinase: effects of ligands

The effects of ligands on the self-association of rabbit muscle phosphofructokinase (PFK) were investigated by velocity sedimentation at pH 7.0 and 23 degrees C. The concentration dependence of the weight-average sedimentation coefficient was monitored in the presence of these ligands. The mode of association and equilibrium constants characterizing each association step were determined by theoretical fitting of the sedimentation data. The simplest mode of association for the PFK system is M in equilibrium M2 equilibrium M4 in equilibrium M16. Ligands and temperature would perturb the various equilibrium constants without altering the mode of association. The apparent equilibrium constants for the formation of tetramer, K4app, are increased in the presence of 0.1 mM ATP and 1.0 mM fructose 6-phosphate. The value of the sedimentation coefficient for the tetramer, S4 degrees, that would best fit the data is 12.4 S instead of 13.5 S determined in the absence of substrates, thus implying a structural change in the tetramer induced by substrates. Only an insignificant amount of dimer is present under the experimental conditions. The presence of activators, ADP or phosphate, enhances the formation of tetramers, and S4 degrees assumes a value of 13.5 S. Similar results are obtained with decreasing concentrations of proton. The presence of the inhibitor, citrate, however, favors the formation of dimers. The equilibrium constants determined as a function of ADP concentration were further analyzed by the linked-function theory derived by Wyman [Wyman, J. (1964) Adv. Protein Chem. 19, 224--285], leading to the conclusion that the formation of a tetramer involves the binding of two additional molecules of ADP per monomer. Similar analysis results in a conclusion that the formation of a dimer involves the binding of one additional molecule of citrate per phosphofructokinase subunit.     

Effects of metabolites on the structural dynamics of rabbit muscle pyruvate kinase

The activity of rabbit muscle pyruvate kinase (PK) is regulated by metabolites. Besides requiring the presence of its substrates, PEP and ADP, the enzyme requires Mg(2+) and K(+) for activity. PK is allosterically inhibited by Phe for activity. The presence of PEP or Phe has opposing effects on the hydrodynamic properties of the enzyme without an apparent change in secondary structure. In this study, the structural perturbation induced by ligand binding was investigated by Fourier transform infrared (FT-IR) spectroscopy. Furthermore, the structural dynamics of PK was probed by H/D exchange monitored by FT-IR. Substrates and activating metal ions induce PK to assume a more dynamic structure while Phe exerts an opposite effect. In all cases there is no significant interconversion of secondary structures. PEP is the most efficient ligand in inducing a change in the microenvironments of both helices and sheets so much so that they can be detected spectroscopically as separate bands. These results provide the first evidence for a differential effect of ligand binding on the dynamics of structural elements in PK. Furthermore, the data support the model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the observed change in hydrodynamic properties represent the two extreme end states.     

Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus,Aeropyrum pernix,and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.     

Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel.

Charybdotoxin (ChTX), a potent peptidyl inhibitor of several types of K+ channels, binds to sites in vascular smooth muscle sarcolemma (Vázquez, J., Feigenbaum, P., Katz, G. M., King, V. F., Reuben, J. P., Roy-Contancin, L., Slaughter, R. S., Kaczorowski, G. J., and Garcia, M. L. (1989) J. Biol. Chem. 265, 20902-20909) which are functionally associated with a high conductance Ca2(+)-activated K+ channel (PK,Ca). 125I-ChTX also binds specifically and reversibly to a single class of sites in plasma membranes prepared from rat brain synaptosomes. These sites exhibit a Kd of 25-30 pM, as measured by either equilibrium or kinetic binding protocols and display a maximum density of about 0.3-0.5 pmol/mg of protein. Competition studies with native ChTX yield a Ki of 8 pM for the noniodinated toxin. The highest density of ChTX sites exists in vesicle fractions of plasma membrane origin. Binding of 125I-ChTX is modulated by metal ions that interact with K+ channels: Ba2+, Ca2+, and Cs+ cause inhibition of ChTX binding; Na+ and K+ stimulate binding at low concentration before producing complete inhibition as their concentration is increased. Stimulation of binding is due to an allosteric interaction that decreases Kd whereas inhibition results from an ionic strength effect. Tetraethylammonium ion has no effect on binding, but tetrabutylammonium ion blocks binding with a Ki of 2.5 mM. Different toxins (i.e. alpha-dendrotoxin, noxiustoxin) that inhibit an inactivating, voltage-dependent K+ channel (PK,V) block 125I-ChTX binding in brain. In marked contrast, iberiotoxin, a selective inhibitor of PK,Ca, has no effect on ChTX binding in this preparation. Inhibition of ChTX binding by alpha-dendrotoxin and noxiustoxin results from an allosteric interaction between separate binding sites for these agents and the ChTX receptor. Taken together, these results suggest that the ChTX sites present in brain are associated with PK,V rather than with PK,Ca. Therefore, 125I-ChTX is a useful probe for elucidating the biochemical properties of a number of different types of K+ channels.     

Investigation of the subunit interactions in malate dehydrogenase.

The dissociations of porcine heart mitochondrial, bovine heart mitochondrial, and porcine heart cytoplasmic malate dehydrogenase dimers (L-malate: NAD+oxidoreductase, EC 1.1.1.37) have been examined by Sephadex G-100 gel filtration chromatography and sedimentation velocity ultracentrifugation. The porcine mitochondrial enzyme was found to chromatograph as subunits when applied to a gel filtration column at a concentration of .02 muM or less at pH 7.0. The presence of coenzymes shifted the dissociation equilibrium at low enzyme concentrations in favor of dimer formation. Monomer formation was also favored when procine mitochondrial enzyme was incubated at pH 5.0 even at concentrations as high as 120 muM. This shift in equilibrium has been correlated with the increased rate and specificity of sulfhydryl residue modification with N-ethylmaleimide at pH 5.0 (Gregory, E.M., Yost, F.J.,Jr., Rohrbach, M.S., and Harrison, J.H. (1971)J. Biol. Chem. 246, 5491-5497). Bovine mitochondrial enzyme did not exhibit a concentration-dependent disociation under the conditions examined. However, at pH5.0 monomer formation was favored, and correlations could again be drawn with sulfhydryl residue modification (Gregory, E.M. (1975)J.Biol. Chem. 250, 5470-5474). In both mitochondrial enzymes, coenzyme binding was found capable of overcoming the effects of pH on the dissociation equilibrium, and dimer formation was favored. Unlike either of the above mentioned enzymes, porcine cytoplasmic malate dehydrogenase did not dissociate into its monomeric form under any conditions investigated.     

Interaction of pyridoxal 5-phosphate with apo-serine hydroxymethyltransferase.

The interaction of pyridoxal 5-phosphate with beef liver serine hydroxymethyltransferase (5,10-methylenetetrahydrofolate:glycine hydroxymethyltransferase, EC 2.1.2.1) has been investigated using sedimentation velocity, kinetic and equilibrium techniques. No evidence for an aggregating system could be found in sedimentation velocity experiments in the presence or absence of pyridoxal 5-phosphate. Reassociation of pyridoxal 5-phosphate with apoenzyme and reacquisition of enzymic activity follow identical kinetics. An initial fast step is followed by a second order process with a rate constant of 66 M-1. s-1. A dissociation constant of 27.5 micrometer was obtained from equilibrium studies. No interaction of binding sites was exposed by altering pH or in the presence of glycine or folate. Maxima observed in pH profiles with both binding and reactivation are interpreted as the composite fo two overlapping processes, one of which is ionization of the pyridinium nitrogen of pyridoxal 5-phosphate and the other a functional group on the apoenzyme. Evidence is presented to indicate the necessity for the formation of an enzyme . pyridoxal 5-phosphate Schiff's base complex during catalytic turnover.     

Energetics of allosteric regulation in muscle pyruvate kinase.

The regulatory mechanism of rabbit muscle pyruvate kinase has been studied as a function of temperature in conjunction with phenylalanine, the allosteric inhibitor. The inhibitory effect of phenylalanine is modulated by temperature. At low temperatures, the presence of phenylalanine is almost inconsequential, but as the temperature increases so does the phenylalanine-dependent inhibition of the kinetic activity. In addition, the presence of phenylalanine induces cooperativity in the relation between velocity and substrate concentration. This effect is especially pronounced at elevated temperature. The kinetic data were analyzed using an equation that describes the steady-state kinetic velocity data as a function of five equilibrium constants and two rate constants. Van't Hoff analysis of the temperature dependence of the equilibrium constants determined by nonlinear curve fitting revealed that the interaction of pyruvate kinase with its substrate, phosphoenolpyruvate, is an enthalpy-driven process. This is consistent with an interaction that involves electrostatic forces, and indeed, phosphoenolpyruvate is a negatively charged substrate. In contrast, the interaction of pyruvate kinase with phenylalanine is strongly entropy driven. These results imply that the binding of phenylalanine involves hydrophobic interaction and are consistent with the basic concepts of strengthening of the hydrophobic effect with an increase in temperature. The effect of phenylalanine at high temperatures is the net consequence of weakening of substrate-enzyme interaction and significant strengthening of inhibitor binding to the inactive state of pyruvate kinase. The effects of salts were also studies. The results show that salts also exert a differential effect on the binding of substrate and inhibitor to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vincristine-induced self-association of calf brain tubulin

The vincristine-induced self-association of tubulin has been examined in a sedimentation velocity study as a function of free drug concentration in PG buffer (0.01 M NaPi and 10(-4) M GTP, pH 7.0) at 20 degrees C. Analysis of the weight-average sedimentation coefficient (S20,w) as a function of protein concentration showed a good fit with the model of an indefinite, isodesmic self-association mechanism. Analysis of the apparent association constants in terms of the Wyman linkage relations showed a good fit to mediation of the self-association by the binding of one ligand molecule. The intrinsic association constant for dimerization of the vincristine-liganded tubulin was found to be 3.8 X 10(5) M-1, and the intrinsic equilibrium constant for the binding of the self-association-linked vincristine molecule had a value of 3.5 X 10(4) M-1, consistent with that measured by fluorescence in our laboratory [Prakash, V., & Timasheff, S. N. (1983) J. Biol. Chem. 258, 1689-1697]. Both reactions are stronger in the presence of vincristine than of vinblastine, reflecting the oxidation of a -CH3 group to -CHO when going from the latter drug to the former one.     

Selective thiol group modification renders fructose-1,6-bisphosphatase insensitive to fructose 2,6-bisphosphate inhibition

Limited treatment of native pig kidney fructose-1,6-bisphosphatase (50 microM enzyme subunit) with [14C]N-ethylmaleimide (100 microM) at 30 degrees C, pH 7.5, in the presence of AMP (200 microM) results in the modification of 1 reactive cysteine residue/enzyme subunit. The N-ethylmaleimide-modified fructose-1,6-bisphosphatase has a functional catalytic site but is no longer inhibited by fructose 2,6-bisphosphate. The enzyme derivative also exhibits decreased affinity toward Mg2+. The presence of fructose 2,6-bisphosphate during the modification protects the enzyme against the loss of fructose 2,6-bisphosphate inhibition. Moreover, the modified enzyme is inhibited by monovalent cations, as previously reported (Reyes, A., Hubert, E., and Slebe, J.C. (1985) Biochem. Biophys. Res. Commun. 127, 373-379), and does not show inhibition by high substrate concentrations. A comparison of the kinetic properties of native and N-ethylmaleimide-modified fructose-1,6-bisphosphatase reveals differences in some properties but none is so striking as the complete loss of fructose 2,6-bisphosphate sensitivity. The results demonstrate that fructose 2,6-bisphosphate interacts with a specific allosteric site on fructose-1,6-bisphosphatase, and they also indicate that high levels of fructose 1,6-bisphosphate inhibit the enzyme by binding to this fructose 2,6-bisphosphate allosteric site.     

Kinetic characterization of the calmodulin-activated catalytic subunit of phosphorylase kinase

The phosphorylase kinase holoenzyme from skeletal muscle is composed of a catalytic and three different regulatory subunits. Analysis of the kinetic mechanism of the holoenzyme is complicated because both the natural substrate phosphorylase b and also phosphorylase kinase itself have allosteric binding sites for adenine nucleotides. In the case of the kinase, these allosteric sites are not on the catalytic subunit. We have investigated the kinetic mechanism of phosphorylase kinase by using its isolated catalytic gamma-subunit (activated by calmodulin) and an alternative peptide substrate (SDQEKRKQISVRGL) corresponding to the convertible region of phosphorylase b, thus eliminating from our system all known allosteric binding sites for nucleotides. This peptide has been previously employed to study the kinetic mechanism of the kinase holoenzyme before the existence of the allosteric sites on the regulatory subunits was suspected [Tabatabai, L. B., & Graves, D. J. (1978) J. Biol. Chem. 253, 2196-2202]. This peptide was determined to be as good an alternative substrate for the isolated catalytic subunit as it was for the holoenzyme. Initial velocity data indicated a sequential kinetic mechanism with apparent Km's for MgATP and peptide of 0.07 and 0.47 mM, respectively. MgADP used as product inhibitor showed competitive inhibition against MgATP and noncompetitive inhibition against peptide, whereas with phosphopeptide as product inhibitor, the inhibition was competitive against both MgATP and peptide. The initial velocity and product inhibition studies were consistent with a rapid equilibrium random mechanism with one abortive complex, enzyme-MgADP-peptide. The substrate-directed, dead-end inhibitors 5'-adenylyl imidodiphosphate and Asp-peptide, in which the convertible Ser of the alternative peptide substrate was replaced with Asp, were competitive inhibitors toward their like substrates and noncompetitive inhibitors toward their unlike substrates, further supporting a random mechanism, which was also the conclusion from the report cited above that used the holoenzyme.     

Structural and regulatory properties of pyruvate kinase from the Cyanobacterium synechococcus PCC 6301

Pyruvate kinase (PK) from the cyanobacterium Synechococcus PCC 6301 was purified 1,300-fold to electrophoretic homogeneity and a final specific activity of 222 micromol of pyruvate produced/min/mg of protein. The enzyme was shown to have a pI of 5.7 and to exist as a 280-kDa homotetramer composed of 66-kDa subunits. This PK appears to be immunologically related to Bacillus PK and a green algal chloroplast PK, but not to rabbit muscle PK, or vascular plant cytosolic and plastidic PKs. The N-terminal amino acid sequence of the Synechococcus PK exhibited maximal (67%) identity with the corresponding region of a putative PK-A sequence deduced from the genome of the cyanobacterium, Synechocystis PCC 6803. Synechococcus PK was relatively heat-labile and displayed a broad pH optimum around pH 7.0. Its activity was not influenced by K(+), but required high concentrations of Mg(2+), and was relatively nonspecific with respect to the nucleoside diphosphate substrate. Potent allosteric regulation by various effectors was observed (activators: hexose monophosphates, ribose 5-phosphate, glycerol 3-phosphate, and AMP; inhibitors: fructose 1,6-bisphosphate, inorganic phosphate, ATP, and several Krebs' cycle intermediates). The enzyme exhibited marked positive cooperativity for phosphoenolpyruvate, which was eliminated or reduced by the presence of the allosteric activators. The results are discussed in terms of the phylogeny and probable central role of PK in the control of cyanobacterial glycolysis.     

MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of Escherichia coli phosphofructokinase

Using enzymatic assays and steady-state fluorescence emission, we performed a linkage analysis of the three-ligand interaction of fructose 6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), and MgATP on E187A mutant Escherichia coli phosphofructokinase (PFK). PEP allosterically inhibits Fru-6-P binding to E. coli PFK. The magnitude of antagonism is 90-fold in the absence and 60-fold in the presence of a saturating concentration of MgATP [Johnson, J. J., and Reinhart, G. D. (1997) Biochemistry 36, 12814-12822]. Substituting an alanine for the glutamate at position 187, located in the allosteric site (i.e., mutant E187A), activates Fru-6-P binding and inhibits the maximal rate of enzyme turnover [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. The allosteric action of PEP appears to depend on the presence of the cosubstrate MgATP. In the presence of a saturating concentration of MgATP, PEP enhances the binding of Fru-6-P to the enzyme by a modest 2-fold. Decreasing the concentration of MgATP mitigates the extent of activation. At MgATP concentrations approaching 25 microM, PEP becomes insensitive to the binding of Fru-6-P. At MgATP concentrations < 25 microM, PEP "crosses over" and becomes antagonistic toward substrate binding. The present study examines the role of Glu 187 at the allosteric site in the binding of Fru-6-P and offers a more complex explanation of the mechanism than that described by traditional allosteric mechanistic models.     

Relation between Mg2+ activation and AMP inhibition of fructose-1,6-diphosphatase from rabbit skeletal muscles

It was shown that AMP, an allosteric inhibitor of fructose-1.6-bisphosphatase, decreases the apparent affinity of the enzyme for the activating cation, Mg2+, which is accompanied by a decrease of the kinetic cooperativity between the Mg2+-binding sites. In its turn, the Mg2+ increase diminishes the enzyme sensitivity to the inhibiting effect of AMP and decreases the cooperativity of the inhibitor binding. The heterotropic interactions between the allosteric inhibitor and activator binding centers are consistent with the predictions of the Monod-Wyman-Changeux model which involves two conformational states of the enzyme (of which one is catalytically inactive) differing in their affinity for the ligands. An increase in pH from 7.4 to 9.0 increases the enzyme affinity for Mg2+ and causes an equilibrium shift towards the catalytically active state of the enzyme.     

Kinetics of ligand binding and quaternary conformational change in the homodimeric hemoglobin from Scapharca inaequivalvis

The kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis has been extensively investigated by flash and dye-laser photolysis, temperature jump relaxation, and stopped flow methods. The results indicate that cooperativity in ligand binding, already observed for oxygen at equilibrium, finds its kinetic counterpart in a large decrease of the oxygen dissociation velocity in the second step of the binding reaction. In the case of carbon monoxide, cooperativity is clearly evident in the increase of the combination velocity constant as the reaction proceeds. Therefore, the ligand-binding kinetics of this dimeric hemoglobin shows the characteristic features of the corresponding reactions of tetrameric hemoglobins. Analysis of the data in terms of the allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) has shown that the values of the allosteric parameters cannot be fixed uniquely for a dimeric hemoglobin. The rapid changes in absorbance observed at the isosbestic points of unliganded and liganded hemoglobin following laser photolysis provided a value of 7 X 10(4) S-1 at 20 degrees C for the rate of the ligand-free quarternary conformational change, postulated on the basis of cooperative ligand binding. Comparison of the rapid absorbance changes observed during ligand rebinding in this hemoglobin with those observed in tuna hemoglobin indicate that, at full photolysis, binding to the T state is followed by further binding and conversion to the liganded R state; at partial photolysis, population of the liganded T state occurs immediately and is followed by a decay to the liganded R state upon further ligand binding. These new results, in conjunction with previous equilibrium data on the same system, show unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas.     

Inhibitor stabilization of human immunodeficiency virus type-2 proteinase dimer formation

We report the first direct observation of the subunit self-association behavior of highly purified recombinant human immunodeficiency virus type-2 (HIV-2) proteinase. Multiple samples of enzyme were subjected to sedimentation equilibrium analytical ultracentrifugation sequentially at 8.8 degrees C and two pH values in the presence and absence of a C2 symmetric, peptidomimetic inhibitor. At both pH values the enzyme exhibited sedimentation equilibrium behavior which fit a monomer-dimer-tetramer model. In the absence of inhibitor, the apparent Kd for dimer formation was less than approximately 100 microM and the apparent Kd for the weaker dimer-tetramer association was greater than approximately 100 microM. In the presence of inhibitor, at either pH, dimer formation was more strongly favored as indicated by a approximately 5-14-fold decrease in the apparent Kd for dimer formation and a approximately 1.2-4-fold increase in the apparent Kd for tetramer formation. The enhanced formation of dimer and decrease in higher order self-associated forms in the presence of an inhibitor is consistent with inhibitor stabilization of an active dimer. The inhibitor-induced stabilization of the dimeric species is consistent with a model for substrate-induced formation of active proteinase dimers in virion assembly.     

Effects of primary sequence differences on the global structure and function of an enzyme: a study of pyruvate kinase isozymes

Pyruvate kinase is an important glycolytic enzyme which is expressed differentially as four distinct isozymes whose catalytic activity is regulated in a tissue-specific manner. The kidney isozyme is known to exhibit sigmoidal kinetics, whereas the muscle isozyme exhibits hyperbolic kinetic properties. By integration of the crystallographic [Stuart, D. I., Levine, M., Muirhead, H., & Stammers, D.K. (1979) J. Mol. Biol. 134, 109-142] and primary sequence data [Noguchi, T., Inoue, H., & Tanaka, T. (1986) J. Biol. Chem. 261, 13807], it was shown that the primary sequence for the C alpha 1 and C alpha 2 regions may constitute the allosteric switching site. To provide insights into the effects of the localized sequence change on the global structural and functional behavior of the enzyme, kinetic studies under a wide spectrum of conditions were conducted for both the muscle and kidney isozymes. These conditions include measurements of enzyme activity as a function of substrate concentrations with different concentrations of allosteric inhibitors or activators. These results showed that both isozymes exhibit the same regulatory properties although quantitatively the distribution of active and inactive forms and the various dissociation constants which govern the binding of substrate and allosteric effectors with the enzyme are different. For such a majority of equilibrium constants to be altered, the localized primary sequence change must confer global perturbations which are manifested as differences in the various equilibrium constants. Structural information about these two isozymes was provided by phase-modulation measurement of the fluorescence lifetime of tryptophan residues under a variety of experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular hydrolysis of formyl peptides and subsequent uptake of liberated amino acids by alveolar macrophages

The mechanism of accumulation of radioactive label from fNle-Leu-[3H]Phe by guinea pig alveolar macrophages was investigated. The binding of fNle-Leu-[3H]Phe to macrophages reached equilibrium within 5 min at 4 degrees C, but equilibrium could not be achieved at temperatures where fNle-Leu-Phe stimulated superoxide anion production is observed (e.g., 21-23 degrees C). At this temperature a rapid phase of initial binding of fNle-Leu-[3H]Phe to its receptor was followed by continued accumulation of cell-associated radioactivity which was linear and was dependent on the extracellular pH, i.e., the rate increased as the pH was lowered from pH 8 to pH 6. Examination for possible intracellular hydrolysis of fNle-Leu-[3H]Phe revealed the presence of extensive amounts of [3H]phenylalanine, both cell-associated and in the medium. The increases in cell-associated [3H]phenylalanine correlated in time and pH with cell-associated radioactivity that was accumulated after stimulation with fNle-Leu-[3H]Phe. The addition of 1 mM unlabelled phenylalanine blocked the long term accumulation of label from fNle-Leu-[3H]Phe by macrophages. 1 mM phenylalanine had no measureable effect on fNle-Leu-Phe stimulated O2- production, fNle-Leu-[3H]Phe hydrolysis or on fNle-Leu-[3H]Phe binding to its receptor. These results indicated that the long term accumulation of radioactivity by alveolar macrophages was due to extracellular hydrolysis of fNle-Leu-[3H]Phe followed by transport of liberated [3H]phenylalanine into the cells. A high affinity (Km = 3.56 X 10(-8) M) transport system for phenylalanine was measured in alveolar macrophages, which was not stimulated by the addition of fNle-Leu-Phe. The extracellular hydrolysis of fNle-Leu-[3H]Phe could not be attributed to release of macrophage enzymes into the medium. The responsible proteinase appears to be membrane bound and has a Km for the hydrolysis of fNle-Leu-[3H]Phe of 2.6 X 10(-7) M which is similar to the Kd (1.5 X 10(-7) M) for fNle-Leu-Phe binding. Taken together, these data suggest that for the alveolar macrophage: (1) formyl peptides are not internalized by a receptor-mediated process; (2) a surface proteinase can catalyze the hydrolysis of formyl peptides; and (3) [3H]phenylalanine formed by fNle-Leu-[3H]Phe hydrolysis is transported into the interior of the macrophage.