Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues

The flavoenzyme UDP-galactopyranose mutase (UGM) is a mediator of cell wall biosynthesis in many pathogenic microorganisms. UGM catalyzes a unique ring contraction reaction that results in the conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UDP-Galf is an essential precursor to the galactofuranose residues found in many different cell wall glycoconjugates. Due to the important consequences of UGM catalysis, structural and biochemical studies are needed to elucidate the mechanism and identify the key residues involved. Here, we report the results of site-directed mutagenesis studies on the absolutely conserved residues in the putative active site cleft. By generating variants of the UGM from Klebsiella pneumoniae, we have identified two arginine residues that play critical catalytic roles (alanine substitution abolishes detectable activity). These residues also have a profound effect on the binding of a fluorescent UDP derivative that inhibits UGM, suggesting that the Arg variants are defective in their ability to bind substrate. One of the residues, Arg280, is located in the putative active site, but, surprisingly, the structural studies conducted to date suggest that Arg174 is not. Molecular dynamics simulations indicate that closed UGM conformations can be accessed in which this residue contacts the pyrophosphoryl group of the UDP-Gal substrates. These results provide strong evidence that the mobile loop, noted in all the reported crystal structures, must move in order for UGM to bind its UDP-galactose substrate.     

Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis.

Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.     

The effects of the side chains of hydrophobic aliphatic amino acid residues in an amphipathic polypeptide on the formation of alpha helix and its association

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.     

Metabolic engineering of Escherichia coli for L-tyrosine production by expression of genes coding for the chorismate mutase domain of the native chorismate mutase-prephenate dehydratase and a cyclohexadienyl dehydrogenase from Zymomonas mobilis

The expression of the feedback inhibition-insensitive enzyme cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase domain from native chorismate mutase-prephenate dehydratase (PheA(CM)) from Escherichia coli was compared to the expression of native feedback inhibition-sensitive chorismate mutase-prephenate dehydrogenase (CM-TyrA(p)) with regard to the capacity to produce l-tyrosine in E. coli strains modified to increase the carbon flow to chorismate. Shake flask experiments showed that TyrC increased the yield of l-tyrosine from glucose (Y(l-Tyr/Glc)) by 6.8-fold compared to the yield obtained with CM-TyrA(p). In bioreactor experiments, a strain expressing both TyrC and PheA(CM) produced 3 g/liter of l-tyrosine with a Y(l-Tyr/Glc) of 66 mg/g. These values are 46 and 48% higher than the values for a strain expressing only TyrC. The results show that the feedback inhibition-insensitive enzymes can be employed for strain development as part of a metabolic engineering strategy for l-tyrosine production.     

Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H⁺, Na⁺, or Li⁺. In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22–100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes. Received: 6 March 2001/Revised: 14 May 2001     

The importance of methionine residues for the catalysis of the biotin enzyme, transcarboxylase. Analysis by site-directed mutagenesis.

Almost all biotin enzymes contain the conserved tetrapeptide Ala-Met-Bct-Met (Bct, N epsilon-biotinyl-L-lysine). In the 1.3 S biotinyl subunit of transcarboxylase (TC), this sequence is present between positions 87 and 90. The conserved nature of these amino acids implies a critical role in the function of biotin enzymes. In order to examine the role of these conserved amino acids, point mutations in the gene encoding the 1.3 S subunit have been made by site-directed mutagenesis to generate A87G, M88L, M90L, M88T, M88C, M88A, and a double mutant A87M, M88A in the 1.3 S subunit. TC, a multisubunit enzyme containing 12 S, 5 S, and 1.3 S subunits, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate (overall reaction). TC can be dissociated into individual subunits and also reconstituted by assembling isolated subunits to a fully active form. The mutants of the 1.3 S subunit have been reconstituted with native 5 S and 12 S subunits from Propionibacterium shermanii. The effects of mutations on the activity of TC were compared with that of TC-1.3 S wild type (WT) prepared in a similar manner. The results show that any substitution of a residue in the conserved tetrapeptide causes impairment of the rate of TC activity. Comparison of gel filtration profiles, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron micrographs of the TC assembled with mutant 1.3 S and with wild type 1.3 S subunits showed that the impairment of the overall activity was not due to a failure of the subunits to assemble into complexes. Steady state kinetic analysis using the mutant 1.3 S subunits indicated that the Km for methylmalonyl-CoA or pyruvate did not change significantly indicating that the binding of substrates is not altered. However, the kcat values were significantly lower for mutants at positions 87 and 88 than for those at position 90. The replacement of methionine at position 88 either by hydrophobic or hydrophilic residues significantly altered the activity in the overall reaction, while similar substitution at position 90 did not dramatically alter the kcat. These results suggest that Ala-87 and Met-88 are catalytically critical in the conserved tetrapeptide.     

The importance of aspartate 327 for catalysis and zinc binding in Escherichia coli alkaline phosphatase.

In order to investigate the function of Asp-327, a bidentate ligand of one of the zinc atoms in Escherichia coli alkaline phosphatase, and the importance of this zinc atom in catalysis, site-specific mutagenesis was used to convert Asp-327 to either asparagine or alanine. The 10(7)-fold decrease in the kcat/Km ratio observed for the Asp-327----Ala enzyme compared to the wild-type enzyme indicates that the side chain of Asp-327 is important for zinc binding at the M1 site. However, only one of the two carboxyl oxygens of Asp-327 is essential for zinc binding, since the Asp-327----Asn enzyme shows approximately the same hydrolysis activity as the wild-type enzyme. The fact that the enzymatic activity of this mutant enzyme shows a dependence on zinc concentration suggests that the other carboxyl oxygen or the negative charge on the side chain of Asp-327 is important in binding of the zinc at the M1 site. However, the zinc hydroxyl must still be appropriately positioned to attack the phosphoserine in the Asp-327----Asn enzyme; therefore, the negative charge and at least one carboxyl oxygen of the side chain are not directly involved in positioning or deprotonating the zinc hydroxyl. 31P NMR studies indicate that the Asp-327----Asn enzyme exhibits transphosphorylation activity at both pH 8.0 and pH 10.0, but at a reduced level compared to the wild-type enzyme. The biphasic production of 2,4-dinitrophenylate in the pre-steady-state kinetics of the mutant enzymes at pH 5.5 suggests that the breaking of the phosphoenzyme covalent complex is rate-limiting for both mutant enzymes. These results suggest that the main function of the zinc atom at the M1 site in catalysis involves decomposition of the phosphoenzyme covalent complex and that it may be important in helping to stabilize the alcohol leaving group.     

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.     

Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181.

A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.     

Induced mutagenesis in dam- mutants of Escherichia coli: a role for 6-methyladenine residues in mutation avoidance.

Summary E. coli strains carrying the dam-3 and dam-4 mutations resulting in reduced levels of 6-methyladenine in the DNA have been found to be more sensitive to base analogue mutagenesis than dam ⁺ strains. Mutagenesis by EMS was also found to be enhanced in dam ^– strains. Dam ^– mutants however were not found to be hypermutable by UV light. It is concluded that the dam ^– strains are deficient in the correct repair of mispairing lesions. The data are consistent with the hypothesis that 6-methyladenine residues in the DNA are involved in strand discrimination during mismatch correction.     

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)     

Conserved and nonconserved residues in the substrate binding site of 7,8-diaminopelargonic acid synthase from Escherichia coli are essential for catalysis

The vitamin B(6)-dependent enzyme 7,8-diaminopelargonic acid (DAPA) synthase catalyzes the antepenultimate step in the synthesis of biotin, the transfer of the alpha-amino group of S-adenosyl-l-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA. The Y17F, Y144F, and D147N mutations in the active site were constructed independently. The k(max)/K(m)(app) values for the half-reaction with DAPA of the Y17F and Y144F mutants are reduced by 1300- and 2900-fold, respectively, compared to the WT enzyme. Crystallographic analyses of these mutants do not show significant changes in the structure of the active site. The kinetic deficiencies, together with a structural model of the enzyme-PLP/DAPA Michaelis complex, point to a role of these two residues in recognition of the DAPA/KAPA substrates and in catalysis. The k(max)/K(m)(app) values for the half-reaction with SAM are similar to that of the WT enzyme, showing that the two tyrosine residues are not involved in this half-reaction. Mutations of the conserved Arg253 uniquely affect the SAM kinetics, thus establishing this position as part of the SAM binding site. The D147N mutant is catalytically inactive in both half-reactions. The structure of this mutant exhibits significant changes in the active site, indicating that this residue plays an important structural role. Of the four residues examined, only Tyr144 and Arg253 are strictly conserved in the available amino acid sequences of DAPA synthases. This enzyme thus provides an illustrative example that active site residues essential for catalysis are not necessarily conserved, i.e., that during evolution alternative solutions for efficient catalysis by the same enzyme arose. Decarboxylated SAM [S-adenosyl-(5')-3-methylthiopropylamine] reacts nearly as well as SAM and cannot be eliminated as a putative in vivo amino donor.     

Nine hydrophobic side chains are key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization domain.

The contribution of almost each amino acid side chain to the thermodynamic stability of the tetramerization domain (residues 326-353) of human p53 has been quantitated using 25 mutants with single-residue truncations to alanine (or glycine). Truncation of either Leu344 or Leu348 buried at the tetramer interface, but not of any other residue, led to the formation of dimers of moderate stability (8-9 kcal/mol of dimer) instead of tetramers. One-third of the substitutions were moderately destabilizing (<3.9 kcal/mol of tetramer). Truncations of Arg333, Asn345 or Glu349 involved in intermonomer hydrogen bonds, Ala347 at the tetramer interface or Thr329 were more destabilizing (4.1-5.7 kcal/mol). Strongly destabilizing (8.8- 11.7 kcal/mol) substitutions included those of Met340 at the tetramer interface and Phe328, Arg337 and Phe338 involved peripherally in the hydrophobic core. Truncation of any of the three residues involved centrally in the hydrophobic core of each primary dimer either prevented folding (Ile332) or allowed folding only at high protein concentration or low temperature (Leu330 and Phe341). Nine hydrophobic residues per monomer constitute critical determinants for the stability and oligomerization status of this p53 domain.     

Effects of multiple replacements at a single position on the folding and stability of dihydrofolate reductase from Escherichia coli

We have made multiple replacements (alanine, arginine, cysteine, histidine, isoleucine, serine, tyrosine) of valine-75 in dihydrofolate reductase from Escherichia coli to examine the relative importance to protein folding of the position that is substituted and the specific character of the amino acid replacement. Valine-75 is part of the eight-stranded beta sheet that forms the structural core of the protein. The isopropyl side chain participates in van der Waals interactions with a number of nonpolar residues, helping to establish a large hydrophobic cluster. Equilibrium studies showed that arginine, histidine, isoleucine, serine, and tyrosine destabilize the protein by 1.9-2.8 kcal mol-1. Alanine and cysteine substitutions have little or no effect. Contrary to other recent studies of the effect of multiple replacements at a hydrophobic site, there is no observed correlation between the changes of the free energy of folding and the changes of the free energy of transfer for the individual amino acids from water to an organic solvent when they are inserted into this site. The effects observed in kinetic studies are both consistent with and extend the equilibrium results; these data indicate that position 75 participates in a rate-limiting step of folding. Some of the equilibrium and kinetic properties of the tyrosine-75 mutant deviated significantly from those of wild-type protein and the other mutants at position 75. (1) The tyrosine variant displayed a complex banding pattern when analyzed by native gel electrophoresis; the wild-type protein and all other mutants at position 75 migrated as single, discrete bands. (2) Comparison of the difference ultraviolet and circular dichroism transition curves showed that a third species is populated at equilibrium; the wild-type protein and all other mutants at position 75 follow a two-state model involving only native and unfolded forms. (3) A third kinetic phase appeared in the unfolding reaction; the wild-type protein and all other mutants at position 75 only showed two kinetic phases in unfolding. Properties 1 and 3 suggest that the tyrosine mutation significantly alters the distribution of native conformers in the protein. These effects on the equilibrium and kinetic data readily display an overriding pattern: residues that would require hydrogen bonding or lead to an expansion of the tightly packed hydrophobic environment in which valine-75 resides destabilize the protein and alter relaxation times of kinetic phases in a consistent manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.     

Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner

Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active‐site residues. However, its catalytic efficiency increases >100‐fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate‐pathway enzyme. The 2.35 Å crystal structure of the MtCM–MtDS complex bound to a transition‐state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active‐site residues on binding to MtDS. The mutagenesis of the C‐terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate‐mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non‐covalent enzyme complexes comprising an AroQ_δ subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.     

The role of tryptophan residues in the function and stability of the mechanosensitive channel MscS from Escherichia coli

Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with phenylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability.     

Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals

Carbamoyl phosphate (CP), the essential precursor of pyrimidines and arginine, is made in Escherichia coli by a single carbamoyl phosphate synthetase (CPS) consisting of 41.4 and 117.7 kDa subunits, which is feed-back inhibited by UMP and activated by IMP and ornithine. The large subunit catalyzes CP synthesis from ammonia in three steps, and binds the effectors in its 15 kDa C-terminal domain. Fifteen site-directed mutations were introduced in 13 residues of this domain to investigate the mechanism of allosteric modulation by UMP and IMP. Two mutations, K993A and V994A, decreased significantly or abolished enzyme activity, apparently by interfering with the step of carbamate synthesis, and one mutation, T974A, negatively affected ornithine activation. S948A, K954A, T974A, K993A and K993W/H995A abolished or greatly hampered IMP activation and UMP inhibition as well as the binding of both effectors, monitored using photoaffinity labeling and ultracentrifugation binding assays. V994A also decreased significantly IMP and UMP binding. L990A, V991A, H995A, G997A and G1008A had more modest effects or affected more the modulation by and the binding of one than of the other nucleotide. K993W, R1020A, R1021A and K1061A were without substantial effects. The results confirm the independence of the regulatory and catalytic centers, and also confirm functional predictions based on the X-ray structure of an IMP-CPS complex. They prove that the inhibitor UMP and the activator IMP bind in the same site, and exclude that the previously observed binding of ornithine and glutamine in this site were relevant for enzyme activation. K993 and V994 appear to be involved in the transmission of the regulatory signals triggered by UMP and IMP binding. These effectors possibly change the position of K993 and V994, and alter the intermolecular contacts mediated by the regulatory domain.