Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation.

Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.     

Kinetics of the interaction of N-(phosphonacetyl)-L-aspartate with the catalytic subunit of aspartate transcarbamoylase. A slow conformational change subsequent to binding

The binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) to the active sites of both the free catalytic subunit of aspartate transcarbamoylase and the intact holoenzyme causes conformational changes which have been studied extensively. However, no kinetic information has been available about the sequence of events occurring during the formation or dissociation of the complexes. Stopped flow kinetics, 31P saturation transfer NMR spectroscopy, and presteady-state kinetics were used to monitor the interaction of PALA with the catalytic subunit (or a derivative containing nitrotyrosyl chromophores which served as spectral probes). The various experimental approaches lead to a mechanism that includes a rapid binding of PALA with an "on" rate of about 10(8)M-1s-1 and an "off" rate of 28 s-1, followed by a much slower isomerization of the complex with a forward rate constant of 0.18 s-1. Analysis of the presteady-state bursts of enzyme activity when the protein is added to a mixture of substrates and PALA and of the lag in activity when the PALA complex with catalytic subunit is added to substrates yielded a rate constant for the reverse isomerization of 0.018s-1. Thus, the conformational change subsequent to PALA binding leads to a 10-fold increase in the equilibrium constant for complex formation. Stopped flow kinetic measurements of the spectral change resulting from mixing the complex of PALA and nitrated protein with native enzyme showed a slow process with a t1/2 of about 11 s, whereas 31P saturation transfer NMR experiments yielded at t1/2 of about 260 ms for the dissociation of PALA from the complex. This apparent disparity is understood in terms of the two-step binding scheme where rapid dissociation of the initial ligand X enzyme complex is measured by the NMR technique and the slow isomerization of the complex is responsible for the bulk of the stopped flow signal.     

Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active site modification, and subunit exchange.

The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined. These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation. Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants. These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored. Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e. the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2). These data point to the interactions between the catalytic center and the subunit interface. Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers. Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers. Hybridization did not occur between avian and mammalian monomers under these conditions. These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface.     

Subunit interactions in yeast glyceraldehyde-3-phosphate dehydrogenase.

The spontaneous inactivation of yeast glyceraldehyde-3-phosphate dehydrogenase was found to fit a simple two-state model at pH 8.5 and 25 degrees. The first step is a relatively rapid dissociation of the tetramer to dimers with the equilibrium largely in favor of the tetramer. In the absence of NAD+ the dimer inactivates irreversibly. The apoenzyme is quite stable with a half-life for complete activity loss proportional to the square root of the enzyme concentration. Perturbances of the protein structure (by pH, ionic strength, and specific salts), which have no effect on the tetrameric state of the molecule, result in an alteration of the cooperativity of NAD+ binding, the reactivity of the active-site sulfhydryl group, and the catalytic activity of the enzyme. Covalent modification of two of the four active-site sulfhydryl groups has profound effects on the enzymic activity which are mediated by changes in the subunit interactions. Sedimentation analysis and hybridization studies indicate that the interaction between subunits remains strong after covalent modification. Under normal physiological and equilibrium dialysis conditions the protein is a tetramer. Equilibrium dialysis studies of NAD+ binding to the enzyme at pH 8.5 and 25 degrees reveal a mixed cooperativity pattern. A model consistent with these observations and the observed half-of-the-sites reactivity is that of ligand induced sequential conformational changes which are transferred across strongly interacting subunit domains. Methods for distinguishing negatively cooperative binding patterns from mixtures of denatured enzyme and multiple species are discussed.     

Proton magnetic relaxation of aspartate transcarbamylase - succinate complexes.

Nuclear magnetic relaxation methods were used to investigate the interaction of the inhibitor succinate with aspartate transcarbamylase from Escherichia coli. Over the pH range 7 to 9, the dissociation constant for succinate remains less than the inhibitor concentration used for most of this work (0.05 M). As a result, the enzyme predominantly exists in a single "gross" conformational state. Succinate binding to this enzyme state (generally known as the R form) parallels the behavior seen previously with the isolated catalytic subunit (Beard, C. B., and Schmidt, P.G. (1973) Biochemistry 12, 2255-2264). The pH and temperature dependence of succinate proton relaxation rates, 1/T2 - 1/T1, in the presence of carbamyl phosphate, is interpreted in terms of a binding mechanism involving three forms of the enzyme, differing by their states of protonation. The least protonated form of the enzyme does not interact with succinate, the singly protonated species binds succinate to form a rapidly dissociating complex, and the doubly protonated species undergoes a conformational isomerization upon succinate binding, yielding a slow exchange complex. Relaxation data provide sufficient information to determine pKa values of 7.2 and 8.9 for two ionizing groups, as well as the dissociation constant for succinate in the fast exchange complex, Kd =1.6 X 10(-2) M. Rate constants for the forward and reverse steps of the isomerization, 1.3 X 10(3) s-1 and 33 s-1, respectively, indicate a significantly slower reverse rate from that obtained in the earlier NMR study of the isolated catalytic subunit. In experiments where the succinate concentration was varied, the relaxation rates showed sigmoidal binding of that ligand to the fast exchange complex above pH 9.1, (a) indicating cooperative binding of succinate, and (b) suggesting that above pH 9.1, the system cannot be characterized by a single dissociation constant, ionization constant, or relaxation effect. CTP and ATP were tested for their ability to affect succinate binding to the fast exchange complex. Heterotropic interactions were observed for CTP but not for ATP. Addition of low concentrations of the transition state analog N-(phosphonacetyl)-L-aspartate to the enzyme-carbamyl phosphate-succinate complex sharply decreased the relaxation rate, indicating that the measurements are sensitive only to succinate bound specifically to the active site.     

Structure of the complex of active site metal-depleted horse liver alcohol dehydrogenase and NADH.

The complex between active site-specific metal-depleted horse liver alcohol dehydrogenase and NADH has been studied with X-ray crystallographic methods to 2.9 A resolution. The electron density maps revealed that only the catalytic zinc ions are removed, whereas the non-catalytic zinc sites ae fully occupied. A gross conformational change in the protein induced by co-enzyme binding takes place in this enzyme species despite the absence of the metal ion in the catalytic center. This circumstance is of great importance in the understanding and further analysis of the trigger mechanisms operating during the conformation transition in alcohol dehydrogenase, since the catalytic center is located at the hinge region for a domain rotation in the subunit, and the metal atom is essential for catalysis. The overall protein structure is the same as that of an NADH complex of the native zinc enzyme and the co-enzyme is bound in a similar manner. The local structural changes observed are restricted to the empty metal binding site.     

Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives

The binding of the methylanthraniloyl derivatives of ATP (mant-ATP), ADP (mant-ADP), 2'deoxyATP (mant-2'deoxyATP), and 3'deoxyATP (mant-3'deoxyATP) to the catalytic subunit of protein kinase A was studied to gain insights into the mechanism of nucleotide binding. The binding of the mant nucleotides leads to a large increase in fluorescence energy transfer at 440 nm, allowing direct measurements of nucleotide affinity. The dissociation constant of mant-ADP is identical to that for ADP, while that for mant-ATP is approximately threefold higher than that for ATP. The dissociation constant for mant-3'deoxyATP is approximately fivefold higher than that for 3'deoxyATP while derivatization of 2'deoxyATP does not affect affinity. The time-dependent binding of mant-ATP, mant-2'deoxyATP, and mant-ADP, measured using stopped-flow fluorescence spectroscopy, is best fit to three exponentials. The fast phase is ligand dependent, while the two slower phases are ligand independent. The slower phases are similar but not identical in rate, and have opposite fluorescence amplitudes. Both isomers of mant-ATP are equivalent substrates, as judged by reversed-phase chromatography, although the rate of phosphorylation is approximately 20-fold lower than the natural nucleotide. The kinetic data are consistent with a three-step binding mechanism in which initial association of the nucleotide derivatives produces a highly fluorescent complex. Either one or two conformational changes can occur after the formation of this binary species, but one of the isomerized forms must have low fluorescence compared to the initial binary complex. These data soundly attest to the structural plasticity within the kinase core that may be essential for catalysis. Overall, the mant nucleotides present a useful reporter system for gauging these conformational changes in light of the prevailing three-dimensional models for the enzyme.     

Comparison of van 't Hoff and calorimetrically determined enthalpies of binding of N-phosphonacetyl-L-aspartate to E. coli aspartate transcarbamylase.

A comparison has been made of the values obtained by direct calorimetric measurements and van 't Hoff analysis, under similar conditions, for the enthalpy of binding of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to E. coli aspartate transcarbamylase and its catalytic subunit. In the case of the catalytic subunit, data were obtained at both saturating and non-saturating concentrations of L-Asp, and at two ionic strengths. Despite a 1000-fold difference in protein concentrations, and the obligatory omission of carbamyl phosphate in the calorimetric experiments, the values obtained by the two methods are shown to agree to within 15% when appropriate corrections are made. These results suggest that subunit dissociation is not a significant factor at the low protein concentrations used in the van 't Hoff analysis, and, conversely, that aggregation of the protein is negligible at the high protein concentrations used in the calorimetric experiments. They also imply that, at pH 8.3, the enthalpic difference between the two conformational states of the enzyme which exist in the presence and absence of substrates is less than 2.5 kcal/mol. In addition, the trends in the three sets of data for the catalytic subunit indicate that ionic bonds are involved in binding PALA to the active site, and that non-productive binding by L-Asp is negligible under these experimental conditions.     

An aspartate transcarbamylase lacking catalytic subunit interactions. Study of conformational changes by ultraviolet absorbance and circular dichroism spectroscopy.

A modified form of aspartate transcarbamylase is synthesized by Escherichia coli in the presence of 2-thiouracil which does not exhibit homotropic cooperative interactions between active sites yet retains heterotropic cooperative interactions due to nucleotide binding. The conformational changes induced in the modified enzyme by the binding of different ligands (substrates, substrate analogs, a transition state analog, and nucleotide effectors) were studied using ultraviolet absorbance and circular dichroism difference spectroscopy. Comparison of the results for the modified enzyme and its isolated subunits to those for the native enzyme and its isolated subunits showed that the conformational changes detected by these methods are qualitatively similar in the two enzymes. Comparison of the absorbance difference spectra due to the binding of a transition substrate analog to the intact native or modified enzymes to the corresponding results for the isolated subunits suggested that ligand binding causes an increased exposure to solvent of certain tyrosyl and phenylalanyl residues in the intact enzymes but not in the isolated subunits. This result is consistent with a diminution of subunit contacts due to substrate binding in the course of homotropic interactions in the native enzyme. Such conformational changes, though perhaps necessary for homotropic cooperativity, are not sufficient to cause homotropic cooperativity since the modified enzyme gave identical perturbations. Interactions of the transition state analog, N-(phosphonacetyl)-L-aspartate, with the modified enzyme were studied. Enzyme kinetic data obtained at low aspartate concentrations showed that this transition state analog does not stimulate activity, but rather exhibits the inhibition predicted for the total absence of homotropic cooperative interactions in the modified enzyme. Spectrophotometric titrations of the number of catalytic sites with the transition state analog showed that the modified enzyme and its isolated subunits possess, respectively, four and two high affinity sites for the inhibitor instead of six and three observed in the case of the normal enzyme and its isolated catalytic subunits. These results are correlated with the lower specific enzymatic activities of the modified enzyme and its catalytic subunits compared to the normal corresponding enzymatic species.     

High-pressure investigations of cytochrome P-450 spin and substrate binding equilibria

The effects of high pressure (1-2000 bar) on the spin state and substrate binding equilibria in cytochrome P-450 have been determined. The high-spin (S = 5/2) to low spin (S = 1/2) transition of the ferric hemoprotein was monitored by uv-visible spectroscopy at various substrate concentrations. Increasing hydrostatic pressure on a sample of substrate-bound cytochrome P-450 resulted in a decrease in the high-spin fraction as monitored by a Soret maxima at 391 nm and an increase in the low-spin 417-nm region of the spectrum. These pressure-induced optical changes were totally reversible for all pressures below 800 bar and were found to correspond to simple substrate dissociation from the enzyme. High levels of the normally metabolized substrate, d-camphor, corresponding to a 99.9% saturation of the hemoprotein active site (50 mM Tris-Cl, 100 mM KCl, pH 7.2) completely prevented the pressure-induced high-spin to low-spin transition that is observed at less than saturating substrate concentrations. A gradual increase in the formation of the inactive P-420 form of the cytochrome was noted if the pressure of the sample was increased above 800 bar. These pressure-linked spectral changes were used to determine the microscopic volume change accompanying substrate binding, which was found to be -47.0 +/- 2 ml/mol (pH 7.2) which represents a substantial change for a ligand dissociation reaction. The observed volume change for camphor binding decreases to -30.6 +/- 2 ml/mol at pH 6.0, suggesting the involvement of a linked proton equilibrium. Various substrate analogs of camphor induce varying degrees of low-spin to high-spin shift upon binding to ferric cytochrome P-450 (3). The volume changes for the dissociation of these substrates were very similar to those obtained with camphor. The conformational changes associated with a shift from high- to low-spin ferric iron appear to be small in comparison to the overall macroscopic changes in volume accompanying substrate binding to the enzyme.     

Inhibitors of different structure induce distinguishing conformations in the omega loop,Cys69-Cys96, of mouse acetylcholinesterase

We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Omega loop), Cys(69)-Cys(96), of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the Omega loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the Omega loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors.     

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.     

Studies of the mechanism of action and regulation of cAMP-dependent protein kinase

Magnetic resonance and kinetic studies of the catalytic subunit of a Type II cAMP-dependent protein kinase from bovine heart have established the active complex to be an enzyme-ATP-metal bridge. The metal ion is β,γ coordinated with Δ chirality at the β-phosphorous atom. The binding of a second metal ion at the active site which bridges the enzyme to the three phosphoryl groups of ATP, partially inhibits the reaction. Binding of the metal-ATP substrate to the enzyme occurs in a diffusion-controlled reaction followed by a 40 ° change in the glycosidic torsional angle. This conformational change results from strong interaction of the nucleotide base with the enzyme. NMR studies of four ATP-utilizing enzymes show a correlation between such conformational changes and high nucleotide base specificity. Heptapeptide substrates and substrate analogs bind to the active site of the catalytic subunit at a rate significantly lower than collision frequency indicating conformational selection by the enzyme or a subsequent slow conformational change. NMR studies of the conformation of the enzyme-bound peptide substrates have ruled out α-helical and β-pleated sheet structures. The results of kinetic studies of peptide substrates in which the amino acid sequence was systematically varied were used to rule out the obligatory requirement for all possible β-turn conformations within the heptapeptide although an enzymatic preference for a β_2–5 or β_3–6 turn could not be excluded. Hence if protein kinase has an absolute requirement for a specific secondary structure, then this structure must be a coil. In the enzyme-substrate complex the distance along the reaction coordinate between the γ-P of ATP and the serine oxygen of the peptide substrate (5.3 ± 0.7 Å) allows room for a metaphosphate intermediate. This finding together with kinetic observations as well as the location of the inhibitory metal suggest a dissociative mechanism for protein kinase, although a mechanism with some associative character remains possible. Regulation of protein kinase is accomplished by competition between the regulatory subunit and peptide or protein substrates at the active site of the catalytic subunit. Thus, the regulatory subunit is found by NMR to block the binding of the peptide substrate to the active site of protein kinase but allows the binding of the nucleotide substrate and divalent cations. The dissociation constant of the regulatory subunit from the active site (10^?10m) is increased ~10-fold by phosphorylation and ~10⁴-fold by the binding of cAMP, to a value (10^?5m) which exceeds the intracellular concentration of the R₂C₂ holoenzyme complex (10^?6m). The resulting dissociation of the holoenzyme releases the catalytic subunit, permitting the active site binding of peptide or protein substrates.     

The use of auramine O to study ligand binding and subunit cooperativity of lactate dehydrogenase

The tetrameric molecule of pig skeletal muscle lactate dehydrogenase binds a cationic fluorescent probe, auramine O, at four equal non-interacting sites with a dissociation constant of (1.25 +/- 0.2) X 10(-4) M. Fluorescence of the dye/enzyme mixture is strongly pH-dependent, with a maximum at pH 6.3-6.8. Auramine O-binding sites are located outside the active center of the enzyme. The microenvironment of the bound dye changes upon interaction of lactate dehydrogenase with NAD+, NADH, ADP and pyruvate. The binding of specific ligands induces an increase in fluorescence of auramine O-enzyme complex. This effect was used to determine the dissociation constants of the complexes of lactate dehydrogenase with specific ligands. Pyruvate was demonstrated to bind to the apoenzyme-auramine O complex with a dissociation constant of 5.2 X 10(-4) M. With the use of auramine O, it became possible to reveal subunit interactions within the tetrameric molecule of lactate dehydrogenase. They are manifested in the changes of the microenvironment of a dye-binding site located on one of the subunits induced by the binding of ligands in the active center of a neighboring subunit.     

A potent fluorescent ATP-like inhibitor of cAMP-dependent protein kinase

The fluorescent ATP analogue 8-azido-2'-O-[14C]dansyl-ATP ([ 14C]AD-ATP) was used to probe the ATP-binding site in the catalytic (C) subunit of cAMP-dependent protein kinase. AD-ATP was found to inhibit the phosphotransferase activity of C subunit with extremely high specificity. Complete inhibition was observed when each mol of C subunit was covalently labeled with 1 mol of this fluorescent ATP analogue. The labeling can be accelerated by the presence of Mg2+ or Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), whereas high concentrations of ATP can almost completely protect the enzyme from AD-ATP. Detailed studies indicated that AD-ATP competes with ATP for binding to C subunit. Analysis of the kinetic data gave dissociation constants of 2.9 and 13 microM for AD-ATP and ATP bound to C subunit, respectively. AD-ATP has a fluorescence emission peak at 510 nm in pH 7.0 aqueous buffer containing 25% glycerol. After covalent binding to C subunit this emission peak shifts to 455 nm, which suggests that the label at ATP site is in an endogenous hydrophobic environment. Upon the binding of Mg2+ or Kemptide, the fluorescence of AD-ATP-labeled C subunit can be enhanced by 50 and 45%, respectively. This enhancement suggests that the binding of either the peptide substrate or Mg2+ induces conformational change at the active site of C subunit. Analysis of the fluorescence data shows that the values of Kd for Mg2+ and Kemptide bound to AD-ATP-labeled C subunit are 0.2 mM and 2.1 microM, respectively. The normal procedure for the preparation of the C subunit from the bovine heart muscle has been simplified to require only one-fifth of the usual working time to obtain the homogeneous enzyme with 70% yield from the crude extract.     

Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent.

The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide. Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate. The modification was not accompanied by aggregation or dissociation. The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme. These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding. The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state. Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax. The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Hervé from cells grown in the presence of 2-thiouracil. However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here. Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme.     

Calculating pKa values in the cAMP-dependent protein kinase: the effect of conformational change and ligand binding

The conformational change observed upon ligand binding and phosphorylation for the cAMP-dependent protein kinase (protein kinase A-PKA) is of high importance for the regulation of its activity. We calculate pKa values and net charges for 18 3D structures of PKA in various conformations and liganded states to examine the role of electrostatics in ligand binding and activation. We find that the conformational change of PKA takes place without any significant net proton uptake/release at all pH values, thus indicating that PKA has evolved to reduce any pH-dependent barriers to the conformational motion. We furthermore find that the binding of ligands induces large changes in the net charge of PKA at most pH values, but significantly, we find that the net charge difference at physiological pH is close to zero, thus indicating that the active-site pKa values have been preorganized for substrate binding. We are unable to unequivocally resolve the identity of the groups responsible for determining the pH-activity profile of PKA but speculate that the titration of Lys 168 or the titration of ATP itself could be responsible for the loss of activity at high pH values. Finally, we examine the effect of point mutations on the pKa values of the PKA catalytic residues and find these to be relatively insensitive to both noncharge-altering and charge-altering mutations.     

Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase.

Nuclear magnetic resonance has been used to study the binding of [13C]carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase. Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar. When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme. The change might also be caused by a ring current froma nearby aromatic amino acid residue. From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies. The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz. Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively. The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0. In contrast, the peak for [1-13C] phosphonaceatmide shifts upfield by about 6 Hz upon binding. Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization. The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation. From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0. The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile. A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry.     

Pyridoxal 5'-phosphate, a fluorescent probe in the active site of aspartate transcarbamylase.

Pyridoxal-P reacts specifically with a single lysine residue at the active site of Escherichia coli aspartate transcarbamylase (Greenwell, P., Jewett, S. L., and Stark, G. R. (1973) J. Biol. Chem. 248, 5994-6001). Reduction of the Schiff base with sodium borohydride, succinylation of the remaining lysine residues, and digestion with trypsin result in formation of a single pyridoxyl peptide, which was purified to homogeneity after chromatography on DEAE-cellulose, treatment with alkaline phosphatase, and rechromatography. Amino acid composition and the results of limited sequential degradation showed that this peptide corresponds to residues 62 to 98 in the sequence of Konigsberg and co-workers, and contains 2 residues of lysine (Henderson, L., Roy, D., Martin, D., and Konigsberg, W., personal communication). By similar isolation, a second peptide was obtained from unsuccinylated catalytic subunit, containing only the pyridoxylated lysine, which corresponds to Lys-80. Derivatives of catalytic subunit containing an average of either one, two, or three pyridoxamine-P moieties per trimer have been prepared by reduction. These species, which retain catalytic activity in proportion to their unmodified active sites, were recombined with regulatory subunit to prepare partially modified derivatives of native aspartate transcarbamylase. At pH 8, fluorescence emission bands were observed at 340 nm, due to aromatic amino acids in the protein, and at 395 nm, due to the pyridoxamine-P moiety. Upon excitation at 280 nm energy transfer from protein to pyridoxamine-P was approximately 15%. The properties of the probe were used to study changes accompanying the binding of substrates and inhibitors. The effects of CTP and ATP were small. With the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) or the substrate carbamyl-P, two types of response were observed. Derivatives of catalytic subunit and native enzyme which contain some unmodified sites and hence retain partial catalytic activity gave large increases in fluorescence at 395 nm. However, fully modified inactive derivatives gave much smaller increases. A derivative of native enzyme containing one triply modified and one unmodified catalytic subunit behaved like the other partially modified species. These results indicate that there is communication among the active sites of different catalytic trimers in modified native enzyme, as well as among active sites within the same modified catalytic trimer. The increases in fluorescence result from a red shift of the absorption maximum of the pyridoxamine-P moiety from 315 to 325 nm, which increases the absorbance at the excitation wavelength for fluorescence. At pH 7, the absorption spectrum is already shifted and, consequently, the binding of PALA and carbamyl-P has little effect on the fluorescence. Therefore, the binding of these compounds at pH 8.0 must cause a structural change in the protein, which in turn causes protonation of a group in the modified active sites, altering the spectral properties.