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H G Britton

Publications and source records attributed to H G Britton.

At least 19 recordsLinked to original sources
PubMed1991

Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain.

The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I.

The requirements for binding at the N-acetyl-L-glutamate binding site of carbamoyl phosphate synthetase I were studied by the displacement of the activator from the central enzyme complex by analogs. Two carboxyls are essential and the acetamido group, if linked to the alpha-carbon, enhances binding 5000-fold. The subsite for the delta-carboxyl is mobile with respect to that for the alpha-carboxyl. Mixtures of complementary fragment of acetylglutamate do not bind, indicating a strong 'chelate' effect. Substituents revealed the existence of steric constraints around the delta-carboxyl, the alpha and gamma-carbons, and the whole of the acetamido group. However, phenyl substituents at the beta-carbon did not hamper binding, indicating that substituents at the beta-carbon face the solution. This is consistent with binding of acetylglutamate as the minimum-energy conformer. All analogs binding with high affinity are activators. Some analogs that bind poorly are competitive inhibitors. They appear to bind preferentially to a low-affinity conformation adopted by the site when the products dissociate and the substrates bind. The acetamido group plays no role in the binding of the inhibitors but it is crucial for the binding of the activators, and the high- and low-affinity conformations of the site differ markedly in structural selectivity.

Acetamides↗
PubMed1988

Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation.

The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.

Adenosine Triphosphate↗
PubMed1985

The mechanism of rabbit muscle phosphofructokinase at pH8.

The mechanism of rabbit muscle phosphofructokinase was investigated by measurement of fluxes, isotope trapping and steady-state velocities at pH8 in triethanolamine/HCl buffer with 4 mM free Mg2+. Most observations were made at I0.2. The ratio Flux of fructose 1,6-bisphosphate----fructose 6-phosphate/Flux of fructose 1,6-bisphosphate----ATP at zero ATP concentration increased hyperbolically from unity to about 3.2 as the concentration of fructose 6-phosphate was increased. Similarly, the ratio Flux of fructose 1,6-bisphosphate----ATP/Flux of fructose 1,6-bisphosphate----fructose 6-phosphate at zero fructose 6-phosphate concentration increased from unity to about 1.4 as the concentration of ATP was increased. The addition of substrates must therefore be random, whatever the other aspects of the reaction. Further, from the plateau values of the ratios, it follows that the substrates dissociate very infrequently from the ternary complex and that at a low substrate concentration 72% of the reaction follows the pathway in which ATP adds first to the enzyme. Isotope-trapping studies with [32P]ATP confirmed that ATP can bind first to the enzyme in rate-limiting step and that dissociation of ATP from the ternary complex is slow in relation to the forward reaction. No isotope trapping of [U-14C]-fructose 6-phosphate could be demonstrated. The ratios Flux of ATP----fructose 1,6-bisphosphate/Flux of ATP----ADP measured at zero ADP concentration and the reciprocal of the ratio measured at zero fructose 1,6-bisphosphate concentration did not differ significantly from unity. Calculated values for these ratios based on the kinetics of the reverse reaction and assuming ordered dissociations of products or a ping-pong mechanism gave values very significantly greater than unity. These findings exclude an ordered dissociation or a substantial contribution from a ping-pong mechanism, and it is concluded that the reaction is sequential and that dissociation of products is random. Rate constants were calculated for the steps in the enzyme reaction. The results indicate a considerable degree of co-operativity in the binding between the two substrates. The observations on phosphofructokinase are discussed in relation to methods of measurement and interpretation of flux ratios and in relation to the mechanism of other kinase enzymes.

Adenosine Diphosphate↗
PubMed1983

Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor.

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO-3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is less than 20% of that of the acetylglutamate-activated enzyme whereas Vmax for the ATPase activity is greater than 40% of that with acetylglutamate. Pulse-chase experiments with H14CO-3 show formation of less "active CO2" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20-30 microM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 microM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time. From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATPA is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the gamma-phosphoryl group of ATPB appears to be facilitated.

Adenosine Triphosphatases↗
PubMed1981

Mechanism of activation of bicarbonate ion by mitochondrial carbamoyl-phosphate synthetase: formation of enzyme-bound adenosine diphosphate from the adenosine triphosphate that yields inorganic phosphate.

The mechanism of the reaction catalyzed by rat liver mitochondrial carbamoyl-phosphate synthetase has been studied by using [beta-18O2]ATP and HC18O-3, monitoring the isotopic composition of adenosine triphosphate (ATP) and inorganic phosphate (Pi) by high-resolution 31P NMR spectroscopy. In the presence of both HCO3- and acetylglutamate, the enzyme catalyzes the exchange of oxygen atoms between the beta, gamma bridging and the beta nonbridging positions of ATP. Addition of NH3 stops the exchange, Pi released by the ATPase activity of the enzyme in the absence of NH3 contains one oxygen atom from HC18O3- but there is no incorporation of 18O into ATP. There is no significant incorporation of [14C]ADP or 32Pi into ATP. It is concluded that in the enzyme-ATPA.HCO30.ATPB complex formed in the presence of ATP and HCO3- there is reversible transfer of the gamma-PO3 group of ATPA (the molecule that yields Pi) to HCO3- without dissociation of products. The beta-PO3 of the enzyme-bound ADP that is formed can rotate. Virtually all of the complex appears to be in the form in which ATPA is cleaved, but in the absence of NH3, ATP is reconstituted and dissociates from the complex on at least 75% of the occasions. On the remainder, the carbonyl phosphate is cleaved in an irreversible process that yields Pi and a low-energy form of carbonic acid (probably HCO3-). NH3 reacts rapidly and irreversibly with the complex, and at saturation the rate (greater than 10 times the rate of Pi release in the absence of NH3) is sufficient to prevent dissociation of ATPA. In the absence of HCO3- an enzyme-ATPA.ATPB complex is formed, but cleavage of the bond between beta, gamma bridging oxygen and P gamma of ATPA does not occur.

Adenosine Diphosphate↗
PubMed1979

Mechanism of carbamoyl-phosphate synthetase. Properties of the two binding sites for ATP.

Carbamoyl phosphate synthethase I synthesizes carbamoyl phosphate from ammonia, HCO3- and two molecules of ATP, one of which, ATPA, yields Pi while the other, ATPB, yields the phosphoryl group of carbamoyl phosphate. Pulse-chase experiments with [gamma-32P]ATP without added HCO3- demonstrate separate binding sites for ATPA and ATPB. Bound ATPA dissociates readily from its site (t1/2 approximately 1--2 s) and the Kd is 0.2--0.7 mM. For the ATPB binding site the t1/2 for dissociation is 5--12 s and the Kd approximately 10 mM. Kd for ATPA seems to increase with enzyme concentration whereas Kd for ATPB does not change. HClO4 releases the ATP unchanged from the enzyme . ATPB and enzyme . ATPB . ATPA complexes. In the presence of HCO3-, ATP and N-acetylglutamate, an enzyme . ATPB . HCO3- . ATPA complex is formed. Its formation by the addition of HCO3- to the enzyme . ATPB . ATPA complex appears to involve an initial bimolecular addition reaction followed by an isomerization. Treatment with HClO4 releases Pi from ATPA but ATPB is released unchanged. Spontaneous hydrolysis of ATPA is responsible for the ATPase activity of the enzyme. Thus, a covalent bond may form between HCO3- and ATPA. However, ATPA can dissociate rapidly (t1/2 less than 10 s). The Kd for ATPA is approximately 0.2 mM. ATPB appears unable to dissociate from the enzyme . ATPB . HCO3- . ATPA complex since the t1/2 for dissociation of ATPB from the enzyme is lengthened about five times in the presence of 19 mM HCO3- and at 1 mM ATP. ATPA may also hydrolyse in this complex and be replaced by another molecule of ATP in the absence of exchange of ATPB. However, the ATPA binding site must be occupied to prevent ATPB release. ATPB may be bound in a pocket which becomes inaccessible to the solution when HCO3- and ATPA also bind. In contrast, HCO3- does not inhibit the binding of ATPB to the enzyme. Various intermediate steps in the formation of the enzyme . ATPb . HCO3- . ATPA complex are discussed. Additional evidence is presented that the ATPB binding site is only periodically accessible to ATP in solution and that ATPB in the steady-state reaction binds when the products leave. Since greater than 1.3 mol ATPB and greater than 1.8 mol ATPA bind/mol enzyme dimer, the enzyme monomer may be an active species.

Adenosine Triphosphate↗
PubMed1979

Mechanism of carbamoyl-phosphate synthetase. Binding of ATP by the rat-liver mitochondrial enzyme.

This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.

Adenosine Triphosphate↗
PubMed1978

Use of flux ratio measurements for the determination of the order of addition of substrates and products in enzyme reactions.

1. Methods of determining the order of addition of substrates and dissociation of products by using flux ratios are investigated. Where an enzyme obeys hyperbolic steady-state velocity kinetics it is concluded that it may be particularly useful to compare the measured flux ratios with those calculated from the steady-state velocity parameters. 2. An expression is derived relating the relative contribution of the two pathways in a branched pathway to the flux ratios. 3. The relationship of equilibrium-reaction-rate measurements [Boyer & Silverstein (1963) Acta Chem. Scand. 17, Suppl. 1, S195] to the flux ratios is considered. Equilibrium-reaction rates are shown to be affected both by the addition of substrates and dissociation of products. Methods of analysing the data to distinguish between these events are discussed. 4. Methods of measurement of flux ratios are described, and it is concluded that the non-equilibrium steady-state method is preferable to measurements at chemical equilibrium. 5. The relative significance of flux ratio measurements and steady-state velocity inhibition data is discussed. It is concluded that flux ratios, when taken in conjunction with the inhibition data, provide the least ambiguous information about mechanism.

Enzymes↗
PubMed1978

Kinetics and mechanism of action of muscle pyruvate kinase.

1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg(2+). 2. Methods of preparing [(32)P]phosphoenolpyruvate from [(32)P]P(i) in high yield and determining [(32)P]-phosphoenolpyruvate and [8-(14)C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [(32)P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-(14)C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [(32)P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg(2+), with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-(14)C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-(14)C]-pyruvate or [gamma-(32)P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.

Adenosine Diphosphate↗
PubMed1978

The supply of citrate to the sheep fetus.

In situ placental perfusion has been used to study the role of the sheep placenta in fetal citrate metabolism. The perfusate citrate concentrations rose steadily to values higher than in fetal plasma. This rise was not influenced by intravenous administration of citrate to the ewe. Concentration and electropotential gradients indicate that transplacental passage is unlikely and the rise is probably due to placental synthesis. However, the quantity of citrate supplied to the fetus was calculated to be small compared with the fetal metabolic rate. Comparison with the guinea pig shows close similarity in fetal plasma concentrations and the amount of citrate formed per unit weight of fetus.

Animals↗
PubMed1977

Calculation of steady-state rate equations and the fluxes between substrates and products in enzyme reactions.

1. Two methods are described for deriving the steady-state velocity of an enzyme reaction from a consideration of fluxes between enzyme intermediates. The equivalent-reaction technique, in which enzyme intermediates are systematically eliminated and replaced by equivalent reactions, appears the most generally useful. The methods are applicable to all enzyme mechanisms, including three-substrate and random Bi Bi Ping Pong mechanisms. Solutions are obtained in algebraic form and these are presented for the common random Bi Bi mechanisms. The steady-state quantities of the enzyme intermediates may also be calculated. Additional steps may be introduced into enzyme mechanisms for which the steady-state velocity equation is already known. 2. The calculation of fluxes between substrates and products in three-substrate and random Bi Bi Ping Pong mechanisms is described. 3. It is concluded that the new methods may offer advantages in ease of calculation and in the analysis of the effects of individual steps on the overall reaction. The methods are used to show that an ordered addition of two substrates to an enzyme which is activated by another ligand will not necessarily give hyperbolic steady-state-velocity kinetics or the flux ratios characteristic of an ordered addition, if the dissociation of the ligand from the enzyme is random.

Enzymes↗
PubMed1977

Mechanism of "L"-type pyruvate kinase from rabbit liver. Evidence against phosphoenzyme formation.

The "L"-type pyruvate kinase from rabbit liver does not catalyse exchange between phosphoenol[1-14C]pyruvate and pyruvate at either pH 8.5 or 6.2. Spectrophotometric experiments at pH 8.5 and 6;2 and gel-filtration experiments with [32P]phosphoenolpyruvate at pH 8,5 also fail to demonstrate phosphoenzyme formation. It is concluded that it is very unlikely that the enzyme has a phosphoenzyme mechanism.

Animals↗
PubMed1976

A study of the permeability of the guinea pig placenta to citrate using recirculating placental perfusion technique.

The role of the guinea pig placenta in fetal citrate metabolism has been studied by perfusing the placenta in situ. The initial citrate concentrations in the perfusate plasma were lower than in the maternal plasma but rose progressively until, in some cases, they exceeded those in the mother. The rise was uninfluenced by intravenous administration of citrate to the mother. Citrate therefore does not enter the perfusate by simple diffusion from the maternal compartment. It may be synthesised within the placenta. From the rate of accumulation of citrate in the perfusate, the supply to the fetus was calculated to be small compared to the fetal metabolic rate. Citrate added to the perfusate became distributed in a space similar to the extracellular space and no change occurred in maternal citrate concentrations. Thus, the placenta does not appear to metabolise citrate or allow transport to the mother.

Animals↗
PubMed1974

The reaction of diethyl pyrocarbonate with pyruvate kinase.

Diethyl pyrocarbonate inactivates muscle pyruvate kinase with the substitution of 3-4 histidine residues per subunit. Phosphoenolpyruvate, ATP and ADP to a lesser extent, and Mg(2+) and pyruvate to a small extent, protect against inactivation.

Adenosine Diphosphate↗
PubMed1974

The mechanism of phosphoglucomutase from Micrococcus lysodeikticus.

The mechanism of the phosphoglucomutase from Micrococcus lysodeikticus was investigated. Induced-transport tests at low substrate concentrations (0.15mm) showed co-transport of the (32)P label but no induced transport of the (14)C label, which is in quantitative agreement with a phosphoenzyme mechanism with a rapid isomerization of the phosphoenzyme. The results excluded an intramolecular transfer of phosphate and could only have been compatible with a sequential mechanism if the K(m) for glucose 1-phosphate had been over 20 times smaller than the measured value. The results of induced-transport tests at intermediate concentrations (1mm) with both labels agreed quantitatively with a phosphoenzyme mechanism, and induced-transport tests with (14)C-labelled substrates at high concentrations (26mm) indicated that the rate constants for isomerization of the phosphoenzyme must be greater than about 3x10(6)s(-1). Consistent with these findings is the fact that (14)C label exchanged between the substrates twice as rapidly as the (32)P label at chemical equilibrium. Further, since the (14)C label exchanged between the substrates about ten times more rapidly than between the substrates and glucose 1,6-diphosphate, glucose 1,6-diphosphate is not an obligatory intermediate in the interconversion of the substrates. It is concluded that, contrary to previous evidence, the mechanism of the enzyme from M. lysodeikticus is essentially that of the rabbit muscle enzyme. To account for the rapid isomerization of the phosphoenzyme in both cases a mechanism is proposed in which there is no formal isomerization of the phosphoenzyme.

Animals↗