Spectral studies on structure-activity relationships of thromboxane synthase inhibitors

Thromboxane A2 synthase is a cytochrome P450-type enzyme and its interaction with imidazole or pyridine-based inhibitors could be studied by absolute and difference spectroscopy with the solubilized as well as the purified enzyme. Nitrogenous bases shift the 418-nm Soret absorption by 4-6 nm to the red and among them the best inhibitors of enzyme activity showed a stoichiometric binding to the enzyme. The structural and energetic prerequisites for such high binding affinities were primarily the liganding of the basic nitrogen to the hemin but also the attachment of a hydrophobic carboxylic side chain to the active site at an about 1 nm distance from the nitrogen. In addition, the side chain seemed to be oriented almost parallel to the plane of the heme. If this geometry was changed, a decrease in affinity was observed and if the ligand binding was sterically hindered, a spectral shift to a five-coordinated complex absorbing at 390 nm occurred. This is best explained by the displacement of an endogenous oxygen ligand, presumably water, from the sixth coordination position of the heme. From these results it can be concluded that the inhibitors mimic the binding of prostaglandin H2 (PGH2) with its carboxylic group at the carboxyl side chain and the endoperoxide oxygen atom at C9 as previously reported. The methyl side chain of PGH2 does not seem to play a role in the formation of the enzyme-substrate complex.     

Dynamics and Binding Modes of Free cdk2 and its Two Complexes with Inhibitors Studied by Computer Simulations

Abstract

This article presents a molecular dynamics (MD) study of the cdk2 enzyme and its two complexes with the inhibitors isopentenyladenine and roscovitine using the Cornell et al. force field from the AMBER software package. The results show that inserting an inhibitor into the enzyme active site does not considerably change enzyme structure but it seemingly changes the distribution of internal motions. The inhibitor causes differences in the domain motions in free cdk2 and in its complexes. It was found out that repulsion of roscovitine N9 substituent causes conformational change on Lys 33 side chain. Isopentenyladenine forms with Lys 33 side chain terminal amino group a hydrogen bond. It implies that the cavity, where N9 substituent of roscovitine is buried, can adopt larger substituent due to Lys 33 side chain flexibility. The composition of electrostatic and van der Waals interactions between the inhibitor and the enzyme were also calculated along both cdk2/inhibitor MD trajectories together with MM-PB/GBSA analysis. These results show that isopentenyladenine-like inhibitors could be more effective after modifications leading to an increase in their van der Waals contact with the enzyme. We suggest that a way leading to better inhibitors occupying isopentenyladenine binding mode could be: to keep N9 and N7 purine positions free, to keep 3,3-dimethylallylamino group at C6 position, and to add, e.g., benzylamino group at C2 position. The results support the idea that the isopentenyladenine binding mode can be used for cdk2 inhibitors design and that all possibilities to improve this binding mode were not uncovered yet.     

Dynamics and binding modes of free cdk2 and its two complexes with inhibitors studied by computer simulations

This article presents a molecular dynamics (MD) study of the cdk2 enzyme and its two complexes with the inhibitors isopentenyladenine and roscovitine using the Cornell et al. force field from the AMBER software package. The results show that inserting an inhibitor into the enzyme active site does not considerably change enzyme structure but it seemingly changes the distribution of internal motions. The inhibitor causes differences in the domain motions in free cdk2 and in its complexes. It was found out that repulsion of roscovitine N9 substituent causes conformational change on Lys 33 side chain. Isopentenyladenine forms with Lys 33 side chain terminal amino group a hydrogen bond. It implies that the cavity, where N9 substituent of roscovitine is buried, can adopt larger substituent due to Lys 33 side chain flexibility. The composition of electrostatic and van der Waals interactions between the inhibitor and the enzyme were also calculated along both cdk2/inhibitor MD trajectories together with MM-PB/GBSA analysis. These results show that isopentenyladenine-like inhibitors could be more effective after modifications leading to an increase in their van der Waals contact with the enzyme. We suggest that a way leading to better inhibitors occupying isopentenyladenine binding mode could be: to keep N9 and N7 purine positions free, to keep 3,3-dimethylallylamino group at C6 position, and to add, e.g., benzylamino group at C2 position. The results support the idea that the isopentenyladenine binding mode can be used for cdk2 inhibitors design and that all possibilities to improve this binding mode were not uncovered yet.     

Structural requirements for specific inhibition of microsomal aminopeptidase by mercaptoamines

L-Leucinthiol, a synthetic derivative of mercaptoethylamine with a hydrophobic side chain, was recently reported to be a potent inhibitor of microsomal aminopeptidase. The structural features necessary for interaction of mercaptoamines with this enzyme have now been explored more systematically. Optimal binding requires a primary amine linked to the mercapto group via two carbon atoms. Only a substituent with L-configuration at the 1 position increased the affinity toward the enzyme. The high degree of specificity and other evidence suggest that the mode of binding of these inhibitors is similar to that of substrates. Comparison of leucinthiol with other amino compounds suggest that the mercapto group makes a much greater contribution to the binding than the hydrophobic side chain. L-Leucinthiol is fairly specific for aminopeptidase although some inhibition of thermolysin and carboxypeptidase A is observed.     

Importance of a conserved residue, aspartate-162, for the function of Escherichia coli aspartate transcarbamoylase.

Aspartate-162 in the catalytic chain of aspartate transcarbamoylase is conserved in all of the sequences determined to date. The X-ray structure of the Escherichia coli enzyme indicates that this residue is located in a loop region (160's loop) that is near the interface between two catalytic trimers and is also close to the active site. In order to test whether this conserved residue is important for support of the internal architecture of the enzyme and/or involved in transmitting homotropic and heterotropic effects, the function of this residue was studied using a mutant version of the enzyme with an alanine at this position (Asp-162----Ala) created by site-specific mutagenesis. The Asp-162----Ala enzyme exhibits a 400-fold reduction in the maximal observed specific activity, approximately 2-fold and 10-fold decreases in the aspartate and carbamoyl phosphate concentrations at half the maximal observed specific activity respectively, a loss of homotropic cooperativity, and loss of response to the regulatory nucleotides ATP and CTP. Furthermore, equilibrium binding studies indicate that the affinity of the mutant enzyme for CTP is reduced more than 10-fold. The isolated catalytic subunit exhibits a 660-fold reduction in maximal observed specific activity compared to the wild-type catalytic subunit. The Km values for aspartate and carbamoyl phosphate for the Asp-162----Ala catalytic subunit were within 2-fold of the values observed for the wild-type catalytic subunit. Computer simulations of the energy-minimized mutant enzyme indicate that the space once occupied by the side chain of Asp-162 may be filled by other side chains, suggesting that Asp-162 is important for stabilizing the internal architecture of the wild-type enzyme.     

Recognition of a cysteine substrate by E. coli gamma-glutamylcysteine synthetase probed by sulfoximine-based transition-state analogue inhibitors

A series of sulfoximine-based transition-state analogue inhibitors with a varying alkyl side chain was synthesized to probe the recognition of a Cys substrate by E. coli gamma-glutamylcysteine synthetase (gamma-GCS). The sulfoximines with a small alkyl group (H, methyl, ethyl, propyl, butyl and CH2OH) each served as a slow-binding inhibitor, the sulfoximine with an ethyl being by far the most potent inhibitor to cause facile and irreversible enzyme inhibition. As the size of the side chain changed from an ethyl, the inhibition potency markedly decreased to reduce the overall affinity with concomitant loss in the inactivation rate and with facile enzyme reactivation by dilution. The sulfoximine without a side chain inhibited the enzyme with almost the same potency as that of L-buthionine-(SR)-sulfoximine (BSO). The free energy difference calculated from the inhibition constants indicates that the side chain of Cys was recognized by its size through hydrophobic interaction and contributed almost equally or even more than the carboxy group to the overall binding of Cys in the transition state.     

Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center

Dihydroorotase plays a key role in pyrimidine biosynthesis by catalyzing the reversible interconversion of carbamoyl aspartate to dihydroorotate. Here we describe the three-dimensional structure of dihydroorotase from Escherichia coli determined and refined to 1.7 A resolution. Each subunit of the homodimeric enzyme folds into a "TIM" barrel motif with eight strands of parallel beta-sheet flanked on the outer surface by alpha-helices. Unexpectedly, each subunit contains a binuclear zinc center with the metal ions separated by approximately 3.6 A. Lys 102, which is carboxylated, serves as a bridging ligand between the two cations. The more buried or alpha-metal ion in subunit I is surrounded by His 16, His 18, Lys 102, Asp 250, and a solvent molecule (most likely a hydroxide ion) in a trigonal bipyramidal arrangement. The beta-metal ion, which is closer to the solvent, is tetrahedrally ligated by Lys 102, His 139, His 177, and the bridging hydroxide. L-Dihydroorotate is observed bound to subunit I, with its carbonyl oxygen, O4, lying 2.9 A from the beta-metal ion. Important interactions for positioning dihydroorotate into the active site include a salt bridge with the guanidinium group of Arg 20 and various additional electrostatic interactions with both protein backbone and side chain atoms. Strikingly, in subunit II, carbamoyl L-aspartate is observed binding near the binuclear metal center with its carboxylate side chain ligating the two metals and thus displacing the bridging hydroxide ion. From the three-dimensional structures of the enzyme-bound substrate and product, it has been possible to propose a unique catalytic mechanism for dihydroorotase. In the direction of dihydroorotate hydrolysis, the bridging hydroxide attacks the re-face of dihydroorotate with general base assistance by Asp 250. The carbonyl group is polarized for nucleophilic attack by the bridging hydroxide through a direct interaction with the beta-metal ion. During the cyclization of carbamoyl aspartate, Asp 250 initiates the reaction by abstracting a proton from N3 of the substrate. The side chain carboxylate of carbamoyl aspartate is polarized through a direct electrostatic interaction with the binuclear metal center. The ensuing tetrahedral intermediate collapses with C-O bond cleavage and expulsion of the hydroxide which then bridges the binuclear metal center.     

Regulatory kinetics of wheat-germ aspartate transcarbamoylase. Adaptation of the concerted model to account for complex kinetic effects of uridine 5''-monophosphate.

The kinetic effects of the end-product inhibitor UMP on aspartate transcarbamoylase (EC 2.1.3.2) purified to homogeneity from wheat germ were studied. In agreement with an earlier study of the relatively crude enzyme [Yon (1972) Biochem. J. 128, 311-320], the half-saturating concentrations of UMP and of the first substrate, carbamoyl phosphate (but not of the second, L-aspartate), were found to be strongly interdependent. However, the kinetic behaviour of the pure enzyme differed from that of the crude enzyme in several important respects, namely: (a) the apparent affinity for UMP was lower with the pure enzyme; (b) sigmoidicity was absent from plots of initial rate versus carbamoyl phosphate concentration, each at a fixed UMP concentration; (c) sigmoidicity was greatly exaggerated in plots of initial rate versus UMP concentration, each at a fixed carbamoyl phosphate concentration, owing to the occurrence of a slight but definite maximum in each plot at low UMP concentration; (d) there was a relative increase in this maximum in the presence of N-phosphonacetyl-L-aspartate, an inhibitor competitive with carbamoyl phosphate. It is shown that a modified two-conformation concerted-transition model can be used to account for most of these features of the pure enzyme. The model treats carbamoyl phosphate and UMP as antagonistic allosteric ligands binding to alternative conformational states [Monod, Wyman & Changeux (1965) J. Mol. Biol. 12, 88-118], carbamoyl phosphate binding non-exclusively (dissociation constants 20 microM and 85 microM respectively) and UMP binding exclusively (dissociation constant 2.5 microM). The model postulates further that the conformation with lower affinity for carbamoyl phosphate has the higher value of kcat., and that it binds UMP in competition with carbamoyl phosphate. Parameters giving the best fit of experimental data to this model were found by a non-linear least-squares search procedure.     

Interaction of mushroom tyrosinase with aromatic amines, o-diamines and o-aminophenols

3-Amino-L-tyrosine was found to be a substrate of mushroom tyrosinase, contrary to what had previously been reported in the literature. A series of amino derivatives of benzoic acid were tested as substrates and inhibitors of the enzyme. 3-Amino-4-hydroxybenzoic acid, 4-amino-3-hydroxybenzoic acid and 3,4-diaminobenzoic acid were oxidized by this enzyme, as previously reported for Neurospora crassa tyrosinase, but 4-aminobenzoic acid and 3-aminobenzoic acid were not. Interestingly, 3-amino-4-hydroxybenzoic acid was oxidized five times faster than 4-amino-3-hydroxybenzoic acid, confirming the importance of proton transfer from the hydroxyl group at C-4 position. All compounds inhibited the monophenolase activity but their effect on the diphenolase activity was small or negligible. 3-Amino-4-hydroxybenzoic acid was a stronger inhibitor than 4-amino-3-hydroxybenzoic acid, indicating their different binding affinity to the oxy form of the enzyme. Both, however, were weaker inhibitors than 3-amino-L-tyrosine, 4-methoxy-o-phenylenediamine and 3,4-diaminobenzoic acid, which was the strongest inhibitor from among the compounds tested. These results show that the relative positioning of the amino group and the hydroxy group in o-aminophenols with respect to the side chain is important both for binding to the dicopper center and for catalysis.     

Substrate specificity and protonation state of Escherichia coli ornithine transcarbamoylase as determined by pH studies. Binding of carbamoyl phosphate

Binding of carbamoyl phosphate to Escherichia coli ornithine transcarbamoylase and its relation to turnover have been examined as a function of pH under steady-state conditions. The pH profile of the dissociation constant of carbamoyl phosphate (Kiacp) shows that the affinity of the substrate increases as pH decreases. Two ionizing groups are involved in carbamoyl phosphate binding. Protonation of an enzymic group with pKa 9.6 results in productive binding of the substrate with a moderate affinity of Kiacp approximately 30 microM. Protonation of a second group further enhances binding by roughly another order of magnitude. This ionization occurs with a pKa that shifts from less than 6 in the free enzyme to 7.3 in the binary complex. However, tighter binding of carbamoyl phosphate due to this ionization does not contribute to catalysis. The turnover rate (kcat) of the enzyme diminishes in the acidic pH range and is governed by an ionization with a pKa of 7.2. Both the catalytic pKa of 7.2 and the productive binding pKa of 9.6 appear in the pH profile of kcat/KMcp. Together with earlier kinetic results (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761), these data suggest that the step which modulates kcat may occur prior to the binding of the second substrate L-ornithine.     

Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain

Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-[14C]glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of unlabeled acetylglutamate was present during the irradiation and (b) the enzyme was replaced by pyruvate kinase, an enzyme that is not affected by acetylglutamate. The labeling was due to the photoactivation of the chloroacetyl group since there was no labeling under equal conditions with acetyl[14C]glutamate. To localize the binding site, limited proteolysis was used. Trypsin cleaves carbamoyl phosphate synthetase into complementary NH2- and COOH-terminal fragments of about 140 and 20 kDa, respectively [Powers-Lee, S. G., & Corina, K. (1986) J. Biol. Chem. 261, 15349-15352], but only the latter was found to be labeled. Similarly, of the various fragments generated by elastase, only two, of 20 and 120 kDa, contain the COOH terminus [see Powers-Lee and Corina (1986) above] and were found to be labeled. Thus, the binding site for acetylglutamate is within 20 kDa from the COOH terminus. This excludes the possibility that the acetylglutamate binding site evolved from an ancestral substrate site for glutamine: this substrate binds to the small subunit of the Escherichia coli enzyme, which is homologous to the NH2-terminal domain of the rat liver enzyme. Exhaustive tryptic digestion of photolabeled carbamoyl phosphate synthetase yielded a single radioactive peak, suggesting that the labeling is restricted to a single minimal tryptic peptide.     

Mobility of the spin-labeled side chains of some novel antifolate inhibitors in their complexes with dihydrofolate reductase

Four spin-labeled inhibitors of dihydrofolate reductase (DHFR) have been synthesized, each of which has the 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) reporting group at a different distance from the 2,4-diaminopyrimidine moiety by which the inhibitors are anchored and oriented in the active site. Inhibitors in which the TEMPO group is attached by a short side chain are weakly bound to DHFR from bacteria (Streptococcus faecium and Lactobacillus casei), to the bovine enzyme and to recombinant human DHFR. However, binding is sufficiently tight, especially in the ternary complexes with NADPH, for recording of the EPR spectra of the bound ligands. The spectra indicate that when these inhibitors are bound to the enzyme the TEMPO group is highly immobilized with correlation time, tau c, 4-20ns. Inhibitors that have the reporter group attached to the glutamate moiety of methotrexate bind to all four DHFRs more tightly than the inhibitors with shorter side chains by factors of up to 10(6). However, in most complexes formed by the inhibitors with longer side chains immobilization of the TEMPO group is slight (tau c 0.2-4 ns). These results are in general agreement with predictions from X-ray crystallographic results including thermal factors but there are some unanticipated differences between some results for bacterial and eukaryotic enzymes. Three of the splin-labeled inhibitors would provide good probes for distance measurements in and around the active site of mammalian DHFR.     

Pseudomonas aeruginosa aspartate transcarbamoylase. Characterization of its catalytic and regulatory properties

Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.     

A calorimetric study of the binding of S-alkylglutathiones to glutathione S-transferase.

The binding of three competitive glutathione analogue inhibitors (S-alkylglutathione derivatives) to glutathione S-transferase from Schistosoma japonicum, SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15--30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S-alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S-methyl-, S-butyl-, and S-octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Delta G(0) remains invariant as a function of temperature, while Delta H(b) and Delta S(0) both decrease as the temperature increases. The binding of three S-alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.     

Monoamine oxidase inhibition by C4-substituted phthalonitriles

It was recently reported that a series of C5-substituted phthalimides are remarkably potent reversible inhibitors of recombinant human monoamine oxidase (MAO) B. Modeling studies suggested that the phthalimide ring forms numerous polar interactions with the polar region of the MAO-B substrate cavity while the C5 side chain extends to, and interacts via Van der Waals interactions with the hydrophobic regions of the enzyme entrance cavity. Interactions with both cavities appear to be requirements for high affinity binding. In the present study we have examined an analogs series of C4-substituted phthalonitriles as potential human MAO inhibitors. The phthalonitriles were found to be highly potent reversible MAO-B inhibitors with most analogs exhibiting IC(50) values in the low nM range. The phthalonitriles also interacted with human MAO-A, although with lower binding affinities compared to MAO-B. Modeling studies suggest that the high binding affinities of the phthalonitriles to MAO-B may depend, at least in part, on the formation of polar interactions between the nitrile functional groups and the enzyme substrate cavity. Examination of a homologs series of benzonitriles established that the phthalonitrile moiety is more optimal for MAO-B inhibition than the corresponding benzonitrile moiety, and that C3-substituted benzonitriles are better MAO-B inhibitors than C4-substituted benzonitriles. Since elimination of the nitrile functional group yielded compounds with only moderate MAO-B inhibition potencies, it may be concluded that this functional group is privileged for MAO-B inhibition.     

Amino-steroids as inhibitors and probes of the active site of cytochrome P-450scc. Effects on the enzyme from different sources

A series of analogues of cholesterol, each having a primary amine attached to a shortened side chain, were tested for their effects on cytochrome P-450scc from several different sources. Reconstituted enzyme systems using disrupted mitochondria from bovine adrenal and placenta, adult human adrenal and placenta, neonatal human adrenal, and rat adrenal and testis were used to assay for inhibitory effects on the side chain cleavage of cholesterol to pregnenolone. Two of the derivatives tested, 22-amino-23,24-bisnor-5-cholen-3 beta-ol and 23-amino-24-nor-5-cholen-3 beta-ol, were found to be potent inhibitors of this reaction; the derivatives in which the amine was attached closer to or further from the steroid ring, (20 R and S)-20-amino-5-pregnen-3 beta-ol and 24-amino-5-cholen-3 beta-ol, were much weaker inhibitors. In addition, spectral studies with rat adrenal mitochondria and a soluble preparation of human placental cytochrome P-450scc showed that binding of the 22-amine derivative to the enzyme produces difference spectra characteristic of nitrogen bonding to the heme; this indicates that the heme is positioned close to C-22 in the steroid-enzyme complex. These findings on the relative effectiveness of the amino-steroid inhibitors and the type of complex formed are similar to results obtained with purified bovine adrenocortical cytochrome P-450scc. This establishes that the proximity of the substrate binding site and the heme-iron catalytic site is a feature common to the enzyme from several sources and is therefore likely to be a necessary property of the active site structure.     

Crystal structure of human ornithine transcarbamylase complexed with carbamoyl phosphate and L-norvaline at 1.9 A resolution

The crystal structure of human ornithine transcarbamylase (OTCase) complexed with carbamoyl phosphate (CP) and L-norvaline (NOR) has been determined to 1.9-A resolution. There are significant differences in the interactions of CP with the protein, compared with the interactions of the CP moiety of the bisubstrate analogue N-(phosphonoacetyl)-L-ornithine (PALO). The carbonyl plane of CP rotates about 60 degrees compared with the equivalent plane in PALO complexed with OTCase. This positions the side chain of NOR optimally to interact with the carbonyl carbon of CP. The mixed-anhydride oxygen of CP, which is analogous to the methylene group in PALO, interacts with the guanidinium group of Arg-92; the primary carbamoyl nitrogen interacts with the main-chain carbonyl oxygens of Cys-303 and Leu-304, the side chain carbonyl oxygen of Gln-171, and the side chain of Arg-330. The residues that interact with NOR are similar to the residues that interact with the ornithine (ORN) moiety of PALO. The side chain of NOR is well defined and close to the side chain of Cys-303 with the side chains of Leu-163, Leu-200, Met-268, and Pro-305 forming a hydrophobic wall. C-delta of NOR is close to the carbonyl oxygen of Leu-304 (3.56 A), S-gamma atom of Cys-303 (4.19 A), and carbonyl carbon of CP (3.28 A). Even though the N-epsilon atom of ornithine is absent in this structure, the side chain of NOR is positioned to enable the N-epsilon of ornithine to donate a hydrogen to the S-gamma atom of Cys-303 along the reaction pathway. Binding of CP and NOR promotes domain closure to the same degree as PALO, and the active site structure of CP-NOR-enzyme complex is similar to that of the PALO-enzyme complex. The structures of the active sites in the complexes of aspartate transcarbamylase (ATCase) with various substrates or inhibitors are similar to this OTCase structure, consistent with their common evolutionary origin.     

Multiple sites and synergism in the binding of inhibitors to microsomal aminopeptidase

The active site of microsomal aminopeptidase has been probed by studying the inhibition of the enzyme in the simultaneous presence of two ligands. The results have been analyzed with the Yonetani-Theorell plot to quantitate the degree of interaction between the two inhibitors. As expected, the enzyme contains a strong binding site for the alpha-amino group and the hydrophobic side chain of specific substrates. In addition, however, the enzyme can interact with another amine and a second hydrophobic group. Evidence suggests that this extra amine may bind to the zinc in an unprotonated form and that one of the hydrophobic sites is located in the vicinity. Another unexpected finding in this work is a strong synergism between the binding of ammonia and that of zinc ligands such as hydroxamates. This synergism may reflect an induced-fit mechanism that brings the catalytically important zinc atom into the optimal state only in the presence of specific substrates.     

Factors accelerating pyrimidine production in <Emphasis Type="Italic">Deinococcus radiophilus</Emphasis>

In studying the pyrimidine synthesising pathway in Deinococcus radiophilus two instances of anomalous behaviour were observed. One was the strikingly different results obtained for two types of assay for carbamoyl phosphate synthetase. Both depend on the fixation of ¹⁴C from the substrate bicarbonate to give radioactive products. In the coupled assay the carbamoyl phosphate product of the enzyme is converted to carbamoyl aspartate in the presence of aspartate and aspartate transcarbamoylase. In the direct assay aspartate is omitted from the reaction mixture and the carbamoyl phosphate is converted to urea. It was found that the radioactive counts in the direct assay were about 5% of those measured in the coupled assay. The second anomaly was that omission of glutamine from both assay mixtures had no significant effect on the fixation of radioactive carbon. These results suggested that aspartate amino-N could be the source of nitrogen for glutamine synthesis by a substrate-channelled pathway which delivered glutamine to carbamoyl phosphate synthetase, and that externally added glutamine could not access its binding site on the 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.