Aspartate transcarbamylase of Escherichia coli. Heterogeneity of binding sites for carbamyl phosphate and fluorinated analogs of carbamyl phosphate.

Some preparations of both native aspartate transcarbamylase from Escherichia coli and catalytic subunit have fewer tight binding sites per oligomer for carbamyl-P than the number of catalytic peptide chains. In contrast, the number of sites for the tight-binding inhibitor N-(phosphonacetyl)-L-aspartate does equal the number of catalytic chains in each case. Binding of the labile carbamyl-P was determined using rapid gel filtration, with conversion to stable carbamyl-L-aspartate during collection. Native enzyme (six catalytic chains) obtained from cells grown under the conditions of J.C. Gerhart and H. Holoubek (J. Biol. Chem. (1967) 242, 2886-2892) has 5.4 tight sites for carbamyl-P at pH 8.0 (KD = 9.9 muM), whereas native enzyme from cells grown with higher concentrations of glucose, uracil, and histidine (to yield more enzyme per unit volume of culture) has only 1.9 tight sites at pH 8.0 (KD = 4.6 muM) and only 2.3 tight sites at pH 7.0 (KD = 2.6 muM). At pH 8.0, catalytic subunit (three catalytic chains) obtained from the former native enzyme has 2.2 tight sites for carbamyl-P (KD = 2.4 muM) and the number of sites is 2.3 in the presence of 35 mM succinate, whereas catalytic subunit obtained from the latter native enzyme has 1.8 tight sites (KD = 3.6 muM) in the absence of succinate and 2.3 tight sites in its presence. The number of tight binding sites is also less than the number of subunit peptide chains in 19F nuclear magnetic resonance experiments performed with catalytic subunit and two fluorinated analogs of carbamyl-P at comparable concentrations of analogs and active sites. A model is proposed in which incomplete removal of formylmethionine from the NH2 termini of the enzyme under conditions of extreme depression affects affinity for ligands.     

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.     

13C isotope effect studies of Escherichia coli aspartate transcarbamylase in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate.

13C isotope effects have been measured for the aspartate transcarbamylase holoenzyme (ATCase) and catalytic subunit catalyzed reactions in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate (PALA). For holoenzyme-catalyzed reactions in the physiological direction with very low levels of L-aspartate as substrate, or with L-cysteine sulfinate as substrate, or in the reverse direction with carbamyl-L-aspartate and phosphate as substrates, the isotope effect data show a slight dependence on PALA concentration. Under these conditions, PALA first stimulates the rate and then inhibits it at higher concentrations. The observed isotope effect at maximum stimulation by PALA is slightly smaller than in the absence of the analog, but as the PALA concentration is increased to reduce the rate to its original value, the observed isotope effect also increases and approaches the value of the isotope effect determined in the absence of PALA. These data suggest that the kinetic properties of the active enzyme are affected by the number of active sites occupied by PALA, indicating communication between subunits, and a mathematical model is proposed which explains our experimental observations. In contrast to these results with the holoenzyme, isotope effects measured for the reaction catalyzed by the isolated catalytic subunits are not altered in the presence of PALA. Taken together, these data are consistent with the two-state model for the homotropic regulation of ATCase.     

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

Bovine liver glutamate dehydrogenase is known to bind reduced coenzyme at two sites/subunit, one catalytic and one regulatory; ADP competes for the latter site. The enzyme is here shown to be catalytically active with the thionicotinamide analogue of NADPH [( S]NADPH). For native enzyme, ultrafiltration studies revealed that [S]NADPH reversibly occupies about two sites/enzyme subunit in the absence of other ligands; by the addition of ADP, [S]NADPH binding can be limited to one molecule/subunit. The enzyme is irreversibly inactivated by reaction with 4-(iodoacetamido)salicylic acid (ISA) at lysine126 within the 2-oxoglutarate binding site [Holbrook, J.J., Roberts, P.A. & Wallis, R.B. (1973) Biochem. J. 133, 165-171]. ISA-modified enzyme binds 1 molecule [S]NADPH/subunit in the absence of ADP, suggesting that reaction at the substrate site blocks binding at the catalytic, but not at the regulatory site. The fluorescence spectrum of ISA-modified enzyme overlaps the absorption spectrum of [S]NADPH allowing a distance measurement between these sites by resonance energy transfer. [S]NADPH quenches the emission of ISA-modified enzyme, yielding 3.2 nm as the average distance between sites. ADP competes for the [S]NADPH site but does not affect the fluorescence of ISA-modified enzyme, indicating that [S]NADPH quenching is attributable to energy transfer rather than to a conformational change. The 3.2 nm thus represents the distance between the 2-oxoglutarate and reduced coenzyme regulatory sites of glutamate dehydrogenase.     

A method for the separation of hybrids of chromatographically identical oligomeric proteins. Use of 3,4,5,6-tetrahydrophthaloyl groups as a reversible "chromatographic handle".

Hybridization experiments with variants of an oligomeric protein often provide important information regarding subunit structure, function, and interactions. In some systems, however, the variants are so similar electrophoretically and chromatographically that purification of individual hybrids is not feasible. Therefore a method was developed for preparing hybrids by using 3,4,5,6-tetrahydrophthalic anhydride as a reversible acylating agent for protein amino groups. The technique involved acylating about 30% of the amino groups at pH 8 to give a derivative with a markedly altered net charge, formation of the hybrid set with unmodified and modified species, separation of the individual components by ion-exchange chromatography, and finally removal of the tetrahydrophthaloyl groups from the desired hybrid by incubation for about 1 day at pH 6 and room temperature. Experiments with model compounds and two enzymes showed that the anhydride was sepcific for amino groups. The extent of modification of proteins was measured by the spectral change at 250 nm, the loss of free amino groups, and the change in electrophoretic mobility of the polypeptide chains in polyacrylamide gels containing 8 M urea. Deacylation of modified, inactive aldolase and the catalytic subunit of aspartate transcarbamylase led to the restoration of the enzyme activity and electrophoretic mobility of the unmodified proteins. Both intra- and inter-subunit hybrids of aspartate transcarbamylase were prepared and isolated by using the tetrahydrophthaloyl groups as a reversible "chromatographic handle". Prior to deacylation the inter-subunit hybrid containing one acylated and one native catalytic subunit (and negative regulatory sub-units) exhibited no homotropic cooperativity and after deacylation the characteristic allosteric properties of the enzyme were regained. Similarly the ligand-promoted conformational changes associated with the allosteric transition were resotred upon deacylation of the intra-subunit hybrid containing one acylated and two native chains in each catalytic subunit. Criteria are described which must be satisfied if a reversible "chromatographic handle" is to be effective in hybridization experiments and it is shown that, despite some heterogeneity in its reaction with protein amino groups, 3,4,5,6-tetrahydrophthalic anhydride shows considerable promise for studies of oligomeric proteins.     

Communication between polypeptide chains in aspartate transcarbamoylase. Conformational changes at the active sites of unliganded chains resulting from ligand binding to other chains

A largely inactive derivative of the catalytic subunit of Escherichia coli aspartate transcarbamoylase containing trinitrophenyl groups on lysine 83 and 84 was used to study communication between polypeptide chains in the holoenzyme and the isolated catalytic trimers. Addition of native regulatory dimers to the derivative yielded a holoenzyme-like complex of low activity which exhibited sigmoidal kinetics and was inhibited by CTP and activated by ATP. The binding of CTP and ATP to the regulatory subunits caused significant and opposite changes in the absorption spectrum resulting from changes in the environment of the sensitive chromophores at the active sites. In allosteric hybrid molecules containing one native and one trinitrophenylated catalytic subunit, along with native regulatory subunits, the binding of a bisubstrate analog, N-(phosphonacetyl)-L-aspartate, to the native catalytic subunit resulted in a perturbation of the spectrum of the chromophore on the unliganded modified chains. Thus the conformational changes associated with the allosteric transition responsible for both heterotropic and homotropic effects are propagated from the sites of ligand binding to the active sites of unliganded distant chains. In addition to the communication from regulatory chains to catalytic chains and the cross-talk from one catalytic subunit to the other, communication between individual catalytic chains in isolated trimers was also demonstrated. By constructing hybrid trimers containing one trinitrophenylated chain and two native chains, we could detect a change in the environment of the chromophore upon the binding of the bisubstrate analog to the native chains.     

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.     

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.     

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.     

The role of dissimilar subunits of NAD-specific isocitrate dehydrogenase from pig heart. Evaluation using affinity labeling

NAD-specific isocitrate dehydrogenase from pig heart is composed of three dissimilar subunits present in the native enzyme as 2 alpha:1 beta: 1 gamma, with a tetramer being the smallest form of complete enzyme. The role of these subunits has been explored using affinity labeling. Specifically labeled subunits are separated and then recombined with unmodified subunits to form dimers. Recombination of beta or gamma subunits modified by the isocitrate analogues, 3-bromo-2-ketoglutarate and 3,4-didehydro-2-ketoglutarate, with unmodified alpha subunit led to the same activity in the dimer as when unmodified beta or gamma was combined with alpha. Contrastingly, modification of alpha with these isocitrate analogues led to loss in activity either alone or when recombined with beta or gamma. Hence, the isocitrate site on alpha is required for catalytic activity but the isocitrate sites on beta or gamma are not necessary for the activity of the functional dimer. Reaction of isolated subunits with 3-bromo-2-ketoglutarate shows that alpha and the alpha beta dimer are modified at about the same rate as holoenzyme, suggestive of similarity of the isocitrate site in native enzyme and in isolated active entities containing alpha subunit; in contrast, beta and gamma subunits react more slowly. Modification by the 2',3'-dialdehyde derivative of the allosteric effector, ADP, led to loss of activity in reconstituted dimers, independent of which subunit was modified. Reaction of isolated subunits with the dialdehyde derivative of ADP is slow compared to the initial reaction with native enzyme, indicating differences in the effects of ADP on intact enzyme and subunits. The ADP sites on all subunits may thus be important in intersubunit interactions, which in turn modulate catalytic activity.     

Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi: thermostability and 1.8A resolution crystal structure of the catalytic subunit complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate

The Pyrococcus abyssi aspartate transcarbamylase (ATCase) shows a high degree of structural conservation with respect to the well-studied mesophilic Escherichia coli ATCase, including the association of catalytic and regulatory subunits. The adaptation of its catalytic function to high temperature was investigated, using enzyme purified from recombinant E.coli cells. At 90 degrees C, the activity of the trimeric catalytic subunit was shown to be intrinsically thermostable. Significant extrinsic stabilization by phosphate, a product of the reaction, was observed when the temperature was raised to 98 degrees C. Comparison with the holoenzyme showed that association with regulatory subunits further increases thermostability. To provide further insight into the mechanisms of its adaptation to high temperature, the crystal structure of the catalytic subunit liganded with the analogue N-phosphonacetyl-L-aspartate (PALA) was solved to 1.8A resolution and compared to that of the PALA-liganded catalytic subunit from E.coli. Interactions with PALA are strictly conserved. This, together with the similar activation energies calculated for the two proteins, suggests that the reaction mechanism of the P.abyssi catalytic subunit is similar to that of the E.coli subunit. Several structural elements potentially contributing to thermostability were identified: (i) a marked decrease in the number of thermolabile residues; (ii) an increased number of charged residues and a concomitant increase of salt links at the interface between the monomers, as well as the formation of an ion-pair network at the protein surface; (iii) the shortening of three loops and the shortening of the N and C termini. Other known thermostabilizing devices such as increased packing density or reduction of cavity volumes do not appear to contribute to the high thermostability of the P.abyssi enzyme.     

Arginine 54 in the active site of Escherichia coli aspartate transcarbamoylase is critical for catalysis: a site-specific mutagenesis, NMR, and X-ray crystallographic study.

The replacement of Arg-54 by Ala in the active site of Escherichia coli aspartate transcarbamoylase causes a 17,000-fold loss of activity but does not significantly influence the binding of substrates or substrate analogs (Stebbins, J.W., Xu, W., & Kantrowitz, E.R., 1989, Biochemistry 28, 2592-2600). In the X-ray structure of the wild-type enzyme, Arg-54 interacts with both the anhydride oxygen and a phosphate oxygen of carbamoyl phosphate (CP) (Gouaux, J.E. & Lipscomb, W.N., 1988, Proc. Natl. Acad. Sci. USA 85, 4205-4208). The Arg-54-->Ala enzyme was crystallized in the presence of the transition state analog N-phosphonacetyl-L-aspartate (PALA), data were collected to a resolution limit of 2.8 A, and the structure was solved by molecular replacement. The analysis of the refined structure (R factor = 0.18) indicates that the substitution did not cause any significant alterations to the active site, except that the side chain of the arginine was replaced by two water molecules. 31P-NMR studies indicate that the binding of CP to the wild-type catalytic subunit produces an upfield chemical shift that cannot reflect a significant change in the ionization state of the CP but rather indicates that there are perturbations in the electronic environment around the phosphate moiety when CP binds to the enzyme. The pH dependence of this upfield shift for bound CP indicates that the catalytic subunit undergoes a conformational change with a pKa approximately 7.7 upon CP binding. Furthermore, the linewidth of the 31P signal of CP bound to the Arg-54-->Ala enzyme is significantly narrower than that of CP bound to the wild-type catalytic subunit at any pH, although the change in chemical shift for the CP bound to the mutant enzyme is unaltered. 31P-NMR studies of PALA complexed to the wild-type catalytic subunit indicate that the phosphonate group of the bound PALA exists as the dianion at pH 7.0 and 8.8, whereas in the Arg-54-->Ala catalytic subunit the phosphonate group of the bound PALA exists as the monoanion at pH 7.0 and 8.8. Thus, the side chain of Arg-54 is essential for the proper ionization of the phosphonate group of PALA and by analogy the phosphate group in the transition state. These data support the previously proposed proton transfer mechanism, in which a fully ionized phosphate group in the transition state accepts a proton during catalysis.     

Three residues involved in binding and catalysis in the carbamyl phosphate binding site of Escherichia coli aspartate transcarbamylase

Site-directed mutagenesis was used to create four mutant versions of Escherichia coli aspartate transcarbamylase at three positions in the catalytic chain of the enzyme. The location of all the amino acid substitutions was near the carbamyl phosphate binding site as previously determined by X-ray crystallography. Arg-54, which interacts with both the anhydride oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme was approximately 17,000-fold less active than the wild type, although the binding of substrates and substrate analogues was not altered substantially. Arg-105, which interacts with both the carbonyl oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme exhibited an approximate 1000-fold loss of activity, while the activity of catalytic subunit isolated from this mutant enzyme was reduced by 170-fold compared to the wild-type catalytic subunit. The KD of carbamyl phosphate and the inhibition constants for acetyl phosphate and N-(phosphono-acetyl)-L-aspartate (PALA) were increased substantially by this amino acid substitution. Furthermore, this loss in substrate and substrate analogue binding can be correlated with the large increases in the aspartate and carbamyl phosphate concentrations at half of the maximum observed specific activity, [S]0.5. Gln-137, which interacts with the amino group of carbamyl phosphate, was replaced by both asparagine and alanine. The asparagine mutant exhibited only a small reduction in activity while the alanine mutant was approximately 50-fold less active than the wild type. The catalytic subunits of both these mutant enzymes were substantially more active than the corresponding holoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A spectral probe near the subunit catalytic site of glutamine synthetase from Escherichia coli. Reduced pyridoxal 5'-phosphate.enzyme complexes.

In order to label phosphate binding sites, unadenylylated glutamine synthetase from Escherichia coli has been pyridoxylated by reacting the enzyme with pyridoxal 5'-phosphate followed by reduction of the Schiff base with NaBH4. A complete loss in Mg2+-supported activity is associated with the incorporation of 3 eq of pyridoxal-P/subunit of the dodecamer. At this extent of modification, however, the pyridoxylated enzyme exhibits substantial Mn2+-supported activity (with increased Km values for ATP and ADP). The sites of pyridoxylation appear to have equal affinities for pyridoxal-P and to be at the enzyme surface, freely accessible to solvent. At least one of the three covalently bound pyridoxamine 5'-phosphate groups is near the subunit catalytic site and acts as a spectral probe for the interactions of the manganese.enzyme with substrates. A spectral perturbation of covalently attached pyridoxamine-P groups is caused also by specific divalent cations (Mn2+, Mg2+ or Ca2+) binding at the subunit catalytic site (but not while binding to the subunit high affinity, activating Me2+ site). In addition, the feedback inhibitors, AMP, CTP, L-tryptophan, L-alanine, and carbamyl phosphate, perturb protein-bound pyridoxamine-P groups. The spectral perturbations produced by substrate and inhibitor binding are pH-dependent and different in magnitude and maximum wavelength. Adenylylation sites are not major sites of pyridoxylation.     

Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe.

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has not effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subnit in gell electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde

The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.     

An essential residue at the active site of aspartate transcarbamylase.

Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both.     

Rat liver glycine methyltransferase. Cooperative binding of S-adenosylmethionine and loss of cooperativity by removal of a short NH2-terminal segment

Rat liver glycine methyltransferase, a homotetramer, exhibits sigmoidal rate behavior with respect to S-adenosylmethionine (Ogawa, H., and Fujioka, M. (1982) J. Biol. Chem. 257, 3447-3452). The binding experiment shows that the sigmoidicity observed in initial velocity kinetics is explained by the cooperative binding of S-adenosylmethionine to the catalytic sites residing on each subunit. Limited proteolysis of glycine methyltransferase with trypsin in the presence of S-adenosylmethionine yields an enzyme lacking the NH2-terminal 8 residues. The proteolytically modified enzyme retains a tetrameric structure. The truncated enzyme shows no cooperativity with respect to S-adenosylmethionine binding and kinetics. It has values of Vmax and Km for glycine identical to those of the native enzyme, but a 3-fold lower [S]0.5 value for S-adenosylmethionine. The proteolytic modification is without effect on the circular dichroism and fluorescence spectra. Furthermore, the protein fluorescence of the modified enzyme is quenched upon addition of S-adenosylmethionine to the same extent as observed with the native enzyme. These results suggest that a short NH2-terminal segment, which lies outside the active site, is important for communication between subunits.     

Chemical modification as a probe of the topography and reactivity of horse-spleen apoferritin.

In apoferritin, but not in ferritin, 1.0 +/- 0.1 cysteine residue per subunit can be modified. In ferritin 3.3 +/- 0.3 lysine residues and 7.1 +/- 0.7 carboxyl groups per subunit can be modified, whilst the corresponding values for apoferritin are 4.4 +/- 0.4 lysine residues and 11.0 +/- 0.4 carboxyl groups per subunit. Modification of lysine residues which maleic anhydride and carboxyl groups with glycineamide in apoferritin which has been dissociated and denatured in guanidine hydrochloride leads to the introduction of 9.1 +/- 0.5 maleyl groups per subunit and 22.0 +/- 0.9 glycineamide residues per subunit. Whereas unmodified apoferritin subunit can be reassociated from guanidine hydrochloride to apoferritin monomer, the ability of maleylated apoferritin to reassociate is impaired. Apoferritin in which all the carboxyl groups have been blocked with glycineamide cannot be reassociated to apoferritin and exists in solution as stable subunits. The modification of one cysteine residue per subunit, of 3 or 4 lysine residues per subunit or of 7 carboxyl groups per subunit has no effect on the catalytic activity of apoferritin. In contrast the modification of 11 carboxyl groups per subunit completely abolishes the catalytic properties of the protein. We conclude that one or more carboxyl groups are essential for the catalytic activity of horse spleen apoferritin.