The reactivity and function of cysteine residues in rabbit liver aldolase B

Rabbit liver aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) contains 8 SH groups/subunit and no disulfide bonds. In the native enzyme 3 SH groups/subunit are titrable with 5,5'-dithiobis(2-nitrobenzoic) acid (Nbs2), 2,2'-dithiodipyridine and N-ethylmaleimide, whereas p-mercuribenzoate is able to react with 4 thiol groups per subunit. Among the three thiol groups titrable with Nbs2, two react 'fast' with simple second-order kinetics, one reacts 'slow' and for this thiol group saturation kinetics is observed, suggesting a reversible binding of Nbs2 to the enzyme prior to covalent modification. It is shown that this binding most likely occurs via ionic interactions in the region close to the active site. The kinetic differentiation between the two 'fast' reacting groups is possible by kinetic analysis of the release of Nbs residues from the modified enzyme. Modification of all exposed SH groups of aldolase B results in 14-32% loss of enzymatic activity. The complete inactivation of liver aldolase by 1 mM p-mercuribenzoate reported previously (Waud, J.M., Feldman, E. and Schray, K.J. (1981) Arch. Biochem. Biophys. 206, 292-295) is shown to be caused by a nonspecific reaction of this reagent used in large excess. It is concluded that this isoenzyme differs from muscle aldolase in the reactivity of exposed SH groups, the mechanisms of the interaction with modifying agents and also in the effect of SH group modification on the enzymatic activity.     

Aspartate aminotransferase from chicken heart cytosol. Characterization of SH-groups]

Homogeneous aspartate aminotransferase has been prepared from chicken heart cytosol. The purification procedure includes fractionation with NH4-sulfate and with ethanol, chromatography on ion-exchange cellulose DE-32 and on hydroxylapatite. Crystallization of the enyme is described. The enzyme was shown to contain 4 SH-groups per protein subunit of molecular weight 50 000. Two of the SH-groups are fully buried, they can be blocked with thiol reagents only upon denaturation of the protein. One exposed SH-group is readily modified at alkaline pH by iodoacetamide, N-ethymaleimide or tetranitromethane, without any inhibition of enzymic activity; this group readily reacts also with 5,5,-ditthiobis (2-nitrobenzoate) and p-mercuribenzoate. One SH-group is semi-buried: it is inaccessible to the above-mentioned reagents at pH 8, but can be blocked by p-mercuribenzoate at pH about 5. Blocking with p-mercuribenzoate of two SH-groups-the exposed and the semi-buried one-lowers enzymic activity to 70% of the initial value. Syncatalytic modication of a SH-group observed in aspartate aminotransferase from pig heart cytosol does not occur in chicken enzyme.     

Evidence for the proximity of two sulfhydryl groups at the active site of 6-phosphogluconate dehydrogenase

The reaction between 6-phosphogluconate dehydrogenase from Candida utilis and 5,5′-dithiobis(2-nitrobenzoate) results in the inactivation of the enzyme. At pH 6.0 the inactivation can be correlated with the modification of only one SH group per enzyme subunit. The modified SH group can react with another SH group forming an intramolecular disulfide bridge. Since the modified enzymes, either with an SH group modified or with a cystine disulfide bridge, are still able to bind the substrate and the coenzyme, gross conformational changes seem unlikely to have occurred. The results obtained suggest that the SH groups of two cysteine residues are located close to each other in the three-dimensional structure of the active site of the enzyme.     

Properties of apoglutamate synthase and comparison with glutamate dehydrogenase.

Glutamate synthase from Escherichia coli K-12 exhibits NH3-dependent activity. NH3-dependent activity is increased approximately 5-fold in apoglutamate synthase lacking flavin and non-heme iron. Whereas glutamine plus 2-oxoglutarate have the capacity to reoxidize the chemically reduced flavoenzyme, no such reoxidation is obtained with 2-oxoglutarate plus NH3. These results establish that the glutamine- and NH3-dependent syntheses of glutamate occur by different pathways of electron transfer from NADPH. The NH3-dependent activity of native and apoglutamate synthase exhibits similar catalytic properties. Some properties of apoglutamate synthase are similar to those of glutamate dehydrogenase. These properties include pH optima for synthesis and oxidative deamination of glutamate, inactivation by alkylating reagents and p-mercuribenzoate, an enhanced rate of inactivation by alkylating reagents and p-mercuribenzoate at low pH, 2-oxoglutarate protection against inactivation by p-mercuribenzoate, and reactivation of p-mercuribenzoate-treated enzyme by 2-mercaptoethanol. 2-Oxoglutarate protects against alkylation of glutamate synthase by iodo [1-14C]acetamide and reduces incorporation of methyl [1-14C]carboxamide into the small subunit of the enzyme.     

Sulfhydryl groups of glucosamine-6-phosphate isomerase deaminase from Escherichia coli

Glucosamine-6-phosphate isomerase deaminase (2-amino-2-deoxy-d-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) from Escherichia coli is an hexameric homopolymer that contains five half-cystines per chain. The reaction of the native enzyme with 5′,5′-dithiobis-(2-nitrobenzoate) or methyl iodide revealed two reactive SH groups per subunit, whereas a third one reacted only in the presence of denaturants. Two more sulfhydryls appeared when denatured enzyme was treated with dithiothreitol, suggesting the presence of one disulfide bridge per chain. The enzyme having the exposed and reactive SH groups blocked with 5′-thio-2-nitrobenzoate groups was inactive, but the corresponding alkylated derivative was active and retained its homotropic cooperativity toward the substrate, d-glucosamine 6-phosphate, and the allosteric activation by N-acetyl-d-glucosamine 6-phosphate. Studies of SH reactivity in the presence of enzyme ligands showed that a change in the availability of these groups accompanies the allosteric conformational transition. The results obtained show that sulfhydryls are not essential for catalysis or allosteric behavior of glucosamine-6-phosphate deaminase.     

Role of tyrosine residues in mitochondrial aspartate aminotransferase from beef kidney.

Mitochondrial aspartate aminotransferase from beef kidney is 50% inhibited after 2 hr treatment with 2.5 mM tetranitromethane at pH 8. Two tyrosine residues per enzyme protomer (46,000 daltons) are modified by the reagent either in the holoenzyme or in the apoenzyme. In both cases the five SH groups titratable with p-mercuribenzoate are not modified by the reagent. However, with a tetranitromethane concentration higher than 2.5 mM and 10 mM mercaptoethanol, an additional tyrosine residue is nitrated in both holo- and apoenzymes. These results are not affected by the presence in the incubation mixture of the substrates alpha-ketoglutarate and glutamate both at ten times their Km values. Mercaptoethanol does not impair the recombination of native or nitrated apoenzyme with the coenzyme and does not reduce the coenzyme moiety of native or nitrated holoenzyme, but promotes a conformational change in the nitrated holoenzyme which causes inactivation. Hydrosulfite promotes the reduction of the coenzyme moiety of native and nitro holoenzyme resulting in their inactivation, largely in the nitrated form. The recombination of the coenzyme with native or nitrated apoenzyme is not influenced by hydrosulfite.     

Sulfhydryl group reactivity of adenosine 3',5'-monophosphate dependent protein kinase from bovine heart: a probe of holoenzyme structure.

The spectrophotometric titration of SH groups in adenosine 3',5'-monophosphate (cAMP) dependent protein kinase from bovine heart muscle with 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB) is described. The holoenzyme (R2C2) contains 16 SH groups, 12 of which react with DTNB in the native enzyme. The SH groups are distributed 2 per catalytic (C) and 4 per regulatory (R) subunit. The binding of cAMP to the holoenzyme or isolated R subunit prevents the reaction of one SH group per R subunit. Modification of SH groups, however, has only a small effect on cAMP binding to R. Reaction of the C subunit with DTNB results in less than 95% loss of catalytic activity. The kinetics of the DTNB reaction and the reversal of the inactivation process by treatment with dithiothreitol suggest that the inactivation is associated with SH group modification. Inactivation studies with the holoenzyme show that: (1) the R subunit inhibits DTNG inactivation of the C subunit in the absence of cAMP; (2) the rate of inactivation of the dephosphoholoenzyme in the presence of cAMP is considerably faster than that of the free catalytic subunit; and (3) the rate of inactivation of the phosphoholoenzyme in the presence of cAMP is faster than that of the C subunit but slower than the dephosphoholoenzyme. The results are interpreted as evidence for a significant interaction of the R and C subunits in the presence of saturating concentrations of cAMP. This interaction is modulated by the state of phosphorylation of R. To account for the inactivation data, a short-lived ternary complex containing R, C, and cAMP is postulated to be in rapid equilibrium with the subunits.     

Heme reactivity of hemoglobins. Azide and fluoride binding equilibria of free and mercuriated ferri-gamma chains.

The free gamma chains, isolated from human foetal hemoglobin, are stable when oxidized and thus suitable for ligand binding and subunit equilibrium studies. The metaquo-ferri chains, with cysteine-F9 in the free state II ag gamma SH) possess several properties which are different from those of their p-mercuribenzoate derivative (III aq gammaSHgR); these are: stronger binding of a high-field ligand (N3- minus), altered spin equilibrium and an altered subunit equilibrium. A quantitative assessment of the free energy changes associated with all individual steps involved in changing the metaquo chains to their azide derivatives has been made. The results show that the higher apparent reactivity of III ag gammaSH (compared to IIIaq gammaSHgR) for the azide ion is not solely due to compensatory effects arising from differences of subunit dissociation or of spin equilibrium: other process(es) occurring in the ligand binding site have to be considered.     

Soybean (Glycine max) urease: Significance of sulfhydryl groups in urea catalysis

The soybean urease (urea amidohydrolase; EC 3.5.1.5) was investigated to elucidate the presence of sulfhydryl (–SH) groups and their significance in urea catalysis with the help of various –SH group specific reagents. The native urease incubated with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) showed exponential increase in the absorbance, thereby revealing the presence of –SH groups. A total of 34 –SH groups per hexamer enzyme molecule were estimated from the absorption studies which represents nearly six –SH groups per subunit. The time-dependent inactivation of urease with DTNB, p-chloromercuribenzoate (p-CMB), N-ethylmaleimide (NEM) and iodoacetamide (IAM) showed biphasic kinetics, where half of the enzyme activity was lost more rapidly than the other half. This study reveals the presence of two categories of “accessible” –SH groups, one category being more reactive than the other. The inactivation of urease by p-CMB was largely reversed on treatment with cysteine, which might be due to unblocking of –SH group by mercaptide exchange reaction. Finally, when NEM inactivated urease was incubated with sodium fluoride, a time-dependent regain of activity was observed with higher concentrations of fluoride ion.     

Differential response of cysteine residues in pig muscle phosphoglucose isomerase to seven sulfhydryl-modifying reagents

The three cysteine residues per subunit of pig muscle phosphoglucose isomerase show different reactivities toward various sulfhydryl reagents. The organomercurial, p-mercuribenzoate, can titrate two of the sulfhydryl groups under nondenaturing conditions. 2,2'-Dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetamide, methyl 2-pyridyl disulfide, and 2-(2'-pyridylmercapto)mercuri-4-nitrophenol all label only one sulfhydryl group under the same conditions, whereas iodoacetic acid does not react with any of the sulfhydryl groups except when the enzyme is fully denatured. It is concluded, therefore, that charge, rather than steric restraint, is the determining factor for the differences seen in the modification patterns of the enzyme by these reagents. When enzyme was first labeled with 2,2'-dithiodipyridine and subsequently with p-mercuribenzoate, it was found that the latter, in a secondary process, will stoichiometrically react with the anion released by the former after the initial reaction with cysteine. The differences in reactivity of the cysteine residues toward the referred-to reagents have been exploited to specifically modify each of the three individual cysteine residues of pig muscle phosphoglucose isomerase.     

Studies on the effect of reagent and protein charges on reactivity of the β93 sulfhydryl group of human hemoglobin using selected mutations

The reactivity of the β93 sulfhydryl (SH) group of human oxyhemoglobins with the negatively charged 5,5′-dithiobis(2,2′-nitrobenzoate) and the uncharged 2,2′-dithiodipyridine was determined as a function of pH. Selected mutant hemoglobins having increased oxygen affinity and having residue substitutions altering charge near the SH group (Wood, Malmö, Yakima, Kempsey, Andrew-Minneapolis, Osler, and Chesapeake) were compared to hemoglobin (Hb) A. Although both reagents reacted with GSH at the same rate and with the same enthalpies of activation, the rates with Hb were different and the difference showed a pronounced pH dependence. The charged reagent was sensitive to charges near the SH group; a positive charge increased the rate and a negative one decreased the rate. The uncharged reagent which reacted with Hb A with activation enthalpies similar to those for GSH was insensitive to neighboring charges, but was sensitive to tertiary and quaternary structural changes. The rates obtained with the latter reagent did not correlate with oxygen affinity. The evolutionary aspects of the β93 cysteine in relation to structure and function are reviewed.     

Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase

The catalytically active oligomeric form of the larger subunit, A_m, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the A_m preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, $\text{Biochemistry}$ 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by A_m was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg²⁺. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.     

Glyceraldehyde-3-phosphate dehydrogenase of horseshoe crab (Tachypleus tridentatus).

Glyceraldehyde-3-phosphate dehydrogenase [ED 1.2.1.12] was purified from the horseshoe crab, a living fossil, and its properties were examined. 1 The purified enzyme was homogeneous as judged by various tests. The enzyme, like enzymes from other sources, was a tetramer with a subunit molecular weight of 36,000. The kinetic parameters and pH optimum were also similar to those of other enzymes, though the enzyme was more stable against heat and pH denaturations. 2 Analysis of SH groups showed that there were 4 SH groups per subunit, one of which was essential for the enzyme activity and was highly reactive. 3. CD spectra of the enzyme suggested that the enzyme had a very high content of beta-structure (ca. 45 per cent). 4. The horseshoe crab enzyme could form a hybrid in vitro with the rabbit muscle enzymes in concentrated salt solution at acidic pH. 5. There results indicate that the enzyme has overall structural similarity to other enzymes and that the enzyme is highly conserved during a long period of evolution. Some discussions on the structure and activity of the horseshoe crab enzyme are made in comparison with the enzymes from other sources.     

Bilevel disulfide group reduction in the activation of c(4) leaf nicotinamide adenine dinucleotide phosphate-malate dehydrogenase

The time course of thioredoxin-mediated reductive activation of isolated Zea mays nicotinamide adenine dinucleotide phosphatemalate dehydrogenase is highly sigmoidal in nature. We examined the factors affecting these kinetics, including the thiol-disulfide status of unactivated and activated forms of the enzyme. The maximum steady rate of activation was increased, and the length of the lag in activation decreased, as the concentrations of thioredoxin-m, dithiothreitol, and KCl were increased. The lag in activation (sigmoidicity) was eliminated by preincubating the unactivated enzyme with 100 mm 2-mercaptoethanol; this pretreatment did not activate the enzyme. Unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase was found to contain approximately two SH groups per subunit, increasing to about four SH per subunit after pretreatment with 2-mercaptoethanol and six SH per subunit after activation by incubating the enzyme with dithiothreitol. We suggest that reduction of one particular higher redox potential disulfide group in unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase facilitates the subsequent reduction of the critical S-S group (regulatory S-S) necessary to generate the active form of the enzyme.     

The active site of 6-phosphogluconate dehydrogenase: A phosphate binding site and its surroundings

Tetrahedral anions bind to a phosphate binding site of 6-phosphogluconate dehydrogenase from Candida utilis, inhibit the enzyme competitively with the 6-phosphogluconate, decrease the reactivity of the SH groups, and mimic the protective effect of 6-phosphogluconate against some inactivating agents. The reaction of the enzyme with butanedione results in the inactivation of the enzyme associated with the modification of a single arginine residue per subunit. This arginine residue may be involved in the binding of the phosphate to the enzyme. Inactivation of the enzyme, upon reaction with permanganate, appears to be due to the oxidation to cysteic acid of a single cysteine residue per enzyme subunit. The reaction of the enzyme with either periodate or hexachloroplatinate causes the loss of the catalytic activity. This inactivation, due to an affinity labeling, is correlated with the oxidation of two SH groups per subunit to an S-S bridge. Photoinactivation of the enzyme by pyridoxal 5′-phosphate is also restricted to the active site of the enzyme. The lysine and the histidine residues involved in this photoinactivation should thus be in the vicinity of the phosphate binding site.     

Functional interactions between subunits of aspartate aminotransferase: Formation of monoliganded dimers during titration of the apoenzyme by pyridoxal 5′-phosphate

The interaction between apoaspartate aminotransferase and pyridoxal 5′-phosphate at either pH 8.3 (active form of holoenzyme) or pH 5.0 (inactive form) corresponds to a strong quenching of tryptophan fluorescence. The hybrid molecule containing one pyridoxal 5′-phosphate bound per dimer has been prepared both by electrofocusing and by ion exchange chromatography. At both pH values, the fluorescence of the hybrid is 80 to 85% of the arithmetic mean between the fluorescence of the symmetrical holoenzyme and apoenzyme. This is direct evidence of energy transfer from tryptophan residues of the subunit of apoenzyme to the coenzyme of the other subunit.Fluorescence intensity was used to determine the quantity of hybrid holoapoenzyme formed during titration of the apoenzyme by pyridoxal 5′-phosphate. At pH 8.3 a non-linear decrease in the fluorescence is observed, corresponding to 60% of hybrid for the point of half reactivation; this value corresponds to the percentage obtained by electrofocusing (Schlegel & Christen, 1974). At pH 5.0, the decrease in fluorescence is linear during pyridoxal binding; this indicates that at this pH the hybrid is never obtained at detectable concentrations. These results indicate strong interactions between subunits of aspartate aminotransferase corresponding to a weakly negative co-operativity at alkaline pH and a positive cooperativity at acidic pH for the binding of the coenzyme.     

Chemical modification of tryptophanase by chloramine T: a possible involvement of the methionine residue in enzyme activity

Tryptophanase purified from Escherichia coli B/1t7-A was irreversibly inactivated by chloramine T (sodium N-chloro-p-toluenesulfonamide). The mode of inactivation was rather complex and did not follow pseudo-first-order kinetics. The inactivation of the apoenzyme was much faster than that of the holoenzyme. The Km value for the synthetic substrate S-o-nitrophenyl-L-cysteine (SOPC) increased concomitantly with the modification. In contrast, the Km value for the coenzyme, pyridoxal 5'-phosphate (PLP), was not altered. L-Serine, another substrate, and L-alanine, a competitive inhibitor, protected the enzyme from inactivation. Determination of SH groups in the enzyme protein with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of two SH groups per enzyme subunit resulted in a complete inactivation. When the enzyme was subjected to chloramine T-modification following the SH group modification with DTNB, further inactivation was still observed, even after the addition of dithiothreitol. The SH-blocked enzyme preparation thus obtained, however, exhibited less pH dependency of inactivation by chloramine T than that of the native enzyme. The amino acid analysis of the chloramine T-modified enzyme showed that modification of four or five methionine residues among the 16 residues per subunit proceeded concomitantly with the complete inactivation. Modification of the enzyme with chloramine T quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the alpha-proton. These results suggest the possibility that chloramine T modifies not only the SH groups, but also methionine residues important for the catalytic activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductase from pig kidney. Amino acid composition, reactivity of cysteinyl residues, and stereochemistry of D-glyceraldehyde reduction.

Some physical and chemical properties of the monomeric NADP+-dependent aldehyde reductase (previously called TPN-L-hexonate dehydrogenase or D-glucuronate reductase) from pig kidney have been examined. The amino acid composition has been determined. Four of the five thiol groups react with p-mercuribenzoate at pH 7, with no resulting loss of catalytic activity. High concentrations of p-mercuribenzoate cause complete enzyme inhibition, which can be partly reversed by addition of aldehyde reductase is low (9%, estimated from the ellipticity at 208 nm), and 70 to 80% of the tyrosine and tryptophan residues aare buried within the molecule. One molecule of NADPH binds to the enzyme (Kp equal 25 muM), causing a blue shift and enhancement of the coenzyme fluorescence, and suggesting that the environment of the active site is hydrophobic. In the reduction of D-glyceraldehyde, catalyzed by aldehyde reductase, the pro-4R "A" hydrogen of NADPH attacks the re face of the carbonyl group. This stereospecificity is the same as in the reductions of D-glyceraldehyde and acetaldehyde effected by rabbit muscle dehydrogenase and liver alcohol dehydrogenase, respectively.     

The modification of sulfhydryl groups of glutamine synthetase from Bacillus stearothermophilus with 5, 5'-dithiobis(2-nitrobenzoic acid).

The SH groups of glutamine synthetase [EC 6.3.1.2] from Bacillus stearothermophilus were modified with 5, 5'-dithiobis(2-nitrobenzoic acid) in order to determine the number of SH groups in the molecule as well as the effect of the modification on the enzyme activity. Three SH groups per subunit were detected after complete denaturation of the enzyme with 6 M urea, one of which was essential for the enzyme activity in view of its reactivity with 5, 5'-dithiobis(2-nitrobenzoic acid) on addition of MgCl2 with loss of the activity. The CD spectra of the modified enzyme in the near ultraviolet region changed from that of the native enzyme, indicating that aromatic amino acid residues were affected by modification of the SH group. The fluorescence derived from tryptophanyl residue(s) was quenched depending on the extent of modification of the SH group, suggesting that the tryptophanyl residue(s) was located in the proximity of the SH group. The thermostability of the enzyme was remarkably decreased by modification of the SH group.     

Reversible reaction of cyanate with a reactive sulfhydryl group at the glutamine binding site of carbamyl phosphate synthetase.

Carbamyl phosphate synthetase from Escherichia coli reacts stoichiometrically (one to one) with [14C]cyanate to give a 14C-labeled complex which can be isolated by gel filtration. The formation of the complex is prevented if L-glutamine is present or if the enzyme is first reacted with 2-amino-4-oxo-5-chloropentanoic acid, a chloro ketone analog of glutamine which has been shown to react with a specific SH group in the glutamine binding site. The rate of complex formation is increased by ADP and decreased by ATP and HCO3-. The isolated complex is inactive with respect to glutamine-dependent synthetase activity. However, the reaction of cyanate with the enzyme is reversible. The rate of dissociation of the isolated complex is not affected by pH (over the pH range 6-10), is greatly increased by ATP and HCO3-, and is decreased by ADP. The allosteric effectors ornithine and UMP have no effect on either the rate of formation or the rate of dissociation of the complex; however, the apparent affinity of the enzyme for ATP is decreased by UMP and increased by ornithine. The site of reaction of cyanate with carbamyl phosphate synthetase, which is composed of a light and a heavy subunit, is with an SH group in the light subunit to give an S-carbamylcysteine residue. The binding of L-[14C]glutamine to the enzyme and the inhibition of glutamine-dependent synthetase activity by the chloroketone analog are both prevented by the presence of cyanate. The reaction with cyanate is considered to be with the same essential SH group which is located in the glutamine binding site and is alkylated by 2-amino-4-oxo-5-chloropentanoic acid. The bicarbonate-dependent effects of ATP suggest that formation of the activated carbon dioxide intermediate is accompanied by changes in the heavy subunit which functionally alter the properties of the glutamine binding site on the light subunit. The allosteric effects of ornithine and UMP are probably not related to this intersubunit interaction.