Guy Hervé

The stimulation of Escherichia coli aspartate transcarbamylase activity by adenosine triphosphate: Relation with the other regulatory conformational changes; a model

Abstract The process of stimulation of Escherichia coli aspartate transcarbamylase activity by ATP was investigated. The efficiency of the phenomenon increases with the number of phosphate groups bound to adenosine. The pH dependence of the stimulation by ATP and adenylyl methylenediphosphonate indicates that the binding of these nucleotides requires the ionization of their last phosphate acidic group. The aspartate trans-carbamylase activity does not appear to be under the influence of the “energy charge ratio” but rather to depend directly on the ATP concentration. The stimulation decreases when the aspartate concentration increases. Saturating amounts of ATP do not provoke the shift in optimum pH for the catalytic activity which is shown to be associated with the homotropic co-operative interactions between the catalytic sites. This result provides additional evidence that homotropic and heterotropic interactions correspond to different molecular mechanisms. ATP reverses the effect of the feedback inhibitor CTP. A model is proposed to account for the relationship between the process of stimulation by ATP and the other regulatory conformational changes.

Coupling of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase

Abstract Several types of conditions allow the disconnection of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase. A model that includes a concerted gross conformational change corresponding to the homotropic cooperative interactions between the catalytic sites and local “site by site” effects promoted by the effectors accounts for this disconnection as well as for the other known properties of the enzyme. However, the substrate concentration influences the extent of stimulation and feedback inhibition of the catalytic activity by the effectors. This result is explained by assuming that these effectors promote a “primary effect”, which is exerted locally “site by site”, and a “secondary effect”, which is mediated by the substrate. As predicted by the model, relaxed (R) forms of the enzyme show only the primary effect. In addition 2-ThioU-aspartate transcarbamylase, a modified form of the enzyme in which the homotropic cooperative interactions between the catalytic sites are selectively abolished, shows the same heterogeneity in CTP binding sites as normal aspartate transcarbamylase.

A reactor permitting injection and sampling for steady state studies of enzymatic reactions at high pressure: Tests with aspartate transcarbamylase

A high pressure reactor for steady state studies of enzymes is described. It allows injection, stirring, and sampling without release of the pressure (up to at least 400 MPa). Thus, either substrate or enzyme can be injected to initiate an enzyme-catalyzed reaction whose progress can then be followed by measurements on samples taken from the reactor. The dead time of sampling is 10-15 s, which allows reactions with pseudo-first-order rate constants smaller than about 1 min-1 to be monitored. It can be used for any enzymatic reaction; unlike previously described high pressure apparatus, it is not limited to the study of enzymes whose activity can be directly followed by spectrophotometry. The use and reliability of this reactor is demonstrated by tests with aspartate transcarbamylase. The activity of this enzyme is enhanced by pressures of the order of 120 MPa.

In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. 3. Catalytic and regulatory properties of carbamylphosphate synthetase: channeling of carbamylphosphate to aspartate transcarbamylase.

The present work reports direct evidence for the channeling of carbamylphosphate from carbamylphosphate synthetase to aspartate transcarbamylase in the multifunctional protein that catalyzes the two first reactions of the pyrimidine pathway in Saccharomyces cerevisiae. This phenomenon is almost certainly related to the previously reported observation that the apparent in situ catalytic mechanism of aspartate transcarbamylase is altered by the association of this enzyme to carbamylphosphate synthetase. As a prerequisite of this investigation, the in situ catalytic and regulatory properties of carbamylphosphate synthetase were studied in the permeabilized cells of a strain that contains the wild-type multifunctional protein but is devoid of the carbamylphosphate synthetase specific for the arginine pathway.

In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae: I. Catalytic and regulatory properties of aspartate transcarbamylase

A permeabilization procedure was adapted to allow the in situ determination of aspartate transcarbamylase activity in Saccharomyces cerevisiae. Permeabilization is obtained by treating cell suspensions with small amounts of 10% toluene in absolute ethanol. After washing, the cells can be used directly in the enzyme assays. Kinetic studies of aspartate transcarbamylase (EC 2.1.3.2) in such permeabilized cells showed that apparent Km for substrates and Ki for the feedback inhibitor UTP were only slightly different from those reported using partially purified enzyme. The aspartate saturation curve is hyperbolic both in the presence and absence of UTP. The inhibition by this nucleotide is noncompetitive with respect to aspartate, decreasing both the affinity for this substrate and the maximal velocity of the reaction. The saturation curves for both substrates give parallel double reciprocal plots. The inhibition by the products is linear noncompetitive. Succinate, an aspartate analog, provokes competitive and uncompetitive inhibitions toward aspartate and carbamyl phosphate, respectively. The inhibition by phosphonacetate, a carbamyl phosphate analog, is uncompetitive and noncompetitive toward carbamyl phosphate and aspartate, respectively, but pyrophosphate inhibition is competitive toward carbamyl phosphate and noncompetitive toward aspartate. These results, as well as the effect of the transition state analog N-phosphonacetyl-L-aspartate, all exclude a random mechanism for aspartate transcarbamylase. Most of the data suggest an ordered mechanism except the substrates saturation curves, which are indicative of a ping-pong mechanism. Such a discrepancy might be related to some channeling of carbamyl phosphate between carbamyl phosphate synthetase and aspartate transcarbamylase catalytic sites.

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli: I. Primary structure of a pyrI gene encoding a modified regulatory subunit☆

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrI encoding the regulatory polypeptide chain of the enzyme. We now report the sequence of the mutated pyrI and show that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Escherichia coli chromosome. An accompanying paper emphasizes the importance of the carboxy-terminal end of the regulatory chain for the homotropic and heterotropic interactions of aspartate carbamoyltransferase.

Proteolysis of type III collagen by collagenase and cathepsin B under high hydrostatic pressure

The effects of high hydrostatic pressures on the kinetics of hydrolysis of type III collagen from calf skin by collagenase and cathepsin B were studied. Collagen hydrolysates sampled at different time intervals (0-90 min) and at different pressures (0.1-300 MPa) were analysed by reverse-phase high pressure liquid chromatography. The rate of collagen hydrolysis decreased up to 300 MPa for both enzymes. The rate of collagen hydrolysis with cathepsin B was drastically reduced between 0.1 and 100 MPa. Significant differences in the populations of the peptides in cathepsin B hydrolysates were observed in chromatograms corresponding to different pressures. This indicated that some amino acid side-chains were less exposed to cathepsin B recognition on the surface of collagen molecules at high pressures. In contrast, the chromatograms of collagenase hydrolysates showed similar patterns, varying only by the peak heights in chromatograms corresponding to the 0.1-300 MPa pressure range. Pressureinduced decreases of the enzyme-apparent activities suggested that the activation volumes for the reaction of both enzymes were positive.

Pyrimidine pathways enzymes in human tumors of brain and associated tissues: Potentialities for the therapeutic use of N-(Phosphonacetyl-l-aspartate and 1-β-d-arabinofuranosylcytosine

The activities of aspartate transcarbamylase (de novo pyrimidine biosynthesis pathway) and of deoxycytidine kinase as well as deoxycytidine deaminase (salvage pyrimidine biosynthesis pathway) were determined in extracts prepared from 40 brain tumors of different types in comparison with extracts from normal nervous tissues. Aspartate transcarbamylase, which is undetectable in normal brain tissue, is present in all tumor samples and in some cases rises to very high activities. Deoxycytidine kinase activity is present in all tissues but its level is generally higher in tumors. Deoxycytidine deaminase is present in all the tissues which were analyzed, although its activity is lower in some of the tumor samples. 1-beta-D-Arabinofuranosylcytosine is a substrate for both deoxycytidine kinase and deaminase in all the samples used except one. These results suggest some potential for the utilization of 1-beta-D-arabinofuranosylcytosine and N-(phosphonacetyl)-L-aspartate in the treatment of brain tumors.

A microtiter plate assay for aspartate transcarbamylase

A discontinuous, colorimetric method for the assay of aspartate transcarbamylase has been adapted for use with 96-well microtiter plates. The method is based on that of L.M. Prescott and M.E. Jones (1969 Anal. Biochem. 32, 408-419) for the detection of ureido compounds, using monoxime and antipyrine. The enzymatic reaction is carried out in a volume of 150 microliters and is stopped by the addition of 100 microliters of a color mix. After development, the absorbance at 460 nm is directly proportional to the quantity of N-carbamyl-L-aspartate up to at least 0.125 mumol and to the quantity of Escherichia coli aspartate transcarbamylase up to about 7 ng. Kinetic parameters obtained from saturation curves for L-aspartate in 50 mM Tris-acetate, pH 8.0, are indistinguishable from those previously obtained: Vmax = 26,225 mumol h-1 mg-1; S0.5 = 14.7 mmol liter-1; hill constant = 2.5.

In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. 2. Reaction mechanism of aspartate transcarbamylase dissociated from carbamylphosphate synthetase by genetic alteration.

The reaction mechanism of Saccharomyces cerevisiae aspartate transcarbamylase was studied in permeabilized cells of a mutant in which this enzyme is not associated to carbamylphosphate synthetase. The results obtained indicate an ordered mechanism in which carbamylphosphate binds first, followed by aspartate, with dissociation of the products in the order phosphate then carbamylaspartate. Interestingly, this clear-cut mechanism differs from the more complex behavior shown by aspartate transcarbamylase when this enzyme is associated to carbamylphosphate synthetase in wild-type S. cerevisiae (B. Penverne and G. Hervé, Arch. Biochem. Biophys. (1983) 225, 562-575). This difference indicates that the association of the two enzymes within the multienzymatic complex alters the apparent kinetic properties of aspartate transcarbamylase. Such an enzyme-enzyme interaction might be related to the channeling of carbamylphosphate from one catalytic site to the other one.