Substrate antagonism in the kinetic mechanism of E. coli phosphofructokinase-1.

In the presence of its allosteric activator GDP, the major phosphofructokinase-1 from Escherichia coli K12 follows Michaelis—Menten kinetics. The kinetic behavior observed at steady-state using different concentrations of the substrates ATP and fructose-6-phosphate and the pattern of inhibition by the substrate analogs adenylyl-(β,γ-methylene)-diphosphonate and D-arabinose-5-phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose-6-phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20-fold when the other substrate is already bound. The antagonism between ATP and fructose-6-phosphate shows that a negative interaction occurs during the reaction with E. coli phosphofructokinase-1 which must be considered in addition to its allosteric properties.     

Renaturation of the allosteric phosphofructokinase from Escherichia coli

The allosteric phosphofructokinase from Escherichia coli has been renatured after complete unfolding in concentrated guanidine hydrochloride. The enzyme regains both its catalytic and regulatory abilities quantitatively. The kinetics of reactivation are biphasic and are consistent with a two-step mechanism in which a monomolecular reaction precedes a bimolecular one. The presence of ATP during reactivation increases the rate at which phosphofructokinase is renatured; the second order rate constant of the bimolecular step increases from about 10(4) M-1 S-1 in the absence of ATP to about 2 X 10(5) M-1 S-1 in the presence of 1 mM ATP. The other ligands of the enzyme have no effect on reactivation. It is tentatively proposed that a folded monomer is the intermediate species which already possesses a functional ATP-binding site and that the rate-limiting association step is the formation of dimeric species. This interpretation is compatible with the known three-dimensional structure of another bacterial phosphofructokinase, that from Bacillus stearothermophilus.     

In vivo metabolism of calcitriol in the pregnant rabbit doe

The production rate, the disappearance rate and the half life of calcitriol in gravid rabbit does at 24 days of gestation were compared, under unstressed steady state conditions, to those of nonpregnant animals. The contribution of the fetoplacental unit to the circulating levels of fetal calcitriol was also assessed. The calcitriol levels (139.6 +/- 19.9 vs 55.3 +/- 8.8 pmol/l and production rates (113.9 +/- 8.8 vs 59.2 +/- 9.2 pmol/min/Kg) were higher in pregnant than in nonpregnant animals (P less than 0.01). However, clearance rates (1.07 +/- 0.18 vs 1.12 +/- 0.20 ml/min/Kg and circulating half life (442 +/- 49 vs 368 +/- 35 min; NS) were similar in both groups of animals. Fetal levels (62.3 +/- 1.6 pmol/l) and specific activity (11166 +/- 864 dpm/pmol) of calcitriol were lower than those of the respective mothers (P less than 0.005). Taken together these data suggest that, calcitriol is transported through the placenta; and that the fetoplacental unit contributes to the fetal and perhaps to the maternal calcitriol levels.     

Mechanism of renaturation of a large protein, aspartokinase-homoserine dehydrogenase

The renaturation of aspartokinase-homoserine dehydrogenase and of some of its smaller fragments has been investigated after complete unfolding by 6 M guanidine hydrochloride. Fluorescence measurements show that a major folding reaction occurs rapidly (in less than a few seconds) after the protein has been transferred to native conditions and results in the shielding of the tryptophan residues from the aqueous solvent; this step also takes place in the fragments and probably corresponds to the independent folding of different segments along the polypeptide chain. The reappearance of the kinase activity, which is an index of the formation of "native" structure within a single chain, is much slower (a few minutes) and has the following properties: it is involved in a kinetic competition with the formation of aggregates; it has an activation energy of 22 +/- 5 kcal/mol; it is not related to a slow reaction in unfolding and thus probably not controlled by the cis-trans isomerization of X-Pro peptide bonds; its rate is inversely proportional to the solvent viscosity. It seems as if this reaction is limited by the mutual arrangement of the regions that have folded rapidly and independently. It is proposed that the mechanism where a fast folding of domains is followed by a slow pairing of folded domains could be generalized to other long chains composed of several domains; such a slow pairing of folded domains would correspond to a rate-limiting process specific to the renaturation of large proteins. The reappearance of the dehydrogenase activity measures the formation of a dimeric species. The dimerization can occur only after each chain has reached its "native" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.     

Substrate antagonism in the kinetic mechanism of E. coli phosphofructokinase-1

In the presence of its allosteric activator GDP, the major phosphofructokinase-1 from Escherichia coli K12 follows Michaelis—Menten kinetics. The kinetic behavior observed at steady-state using different concentrations of the substrates ATP and fructose-6-phosphate and the pattern of inhibition by the substrate analogs adenylyl-(β,γ-methylene)-diphosphonate and D-arabinose-5-phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose-6-phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20-fold when the other substrate is already bound. The antagonism between ATP and fructose-6-phosphate shows that a negative interaction occurs during the reaction with E. coli phosphofructokinase-1 which must be considered in addition to its allosteric properties.     

A triglobular model for the polypeptide chain of aspartokinase I-homoserine dehydrogenase I of Escherichia coli

Limited proteolysis of aspartokinase I-homoserine dehydrogenase I from Escherichia coli by type VI protease from Streptomyces griseus yields five proteolytic fragments, three of which are dimeric, the other two being monomeric. One of the monomeric fragments (27 kilodaltons) exhibits residual aspartokinase activity, while the second one (33 kilodaltons) possesses residual homoserine dehydrogenase activity. The smallest of the dimeric species (2 X 25 kilodaltons) is inactive; the two other dimers exhibit either only homoserine dehydrogenase activity (2 X 59 kilodaltons) or both activities (hybrid fragment, 89 + 59 kilodaltons). This characterization of the proteolytic species in terms of molecular weight, subunit structure, and activity leads to the proposal of a triglobular model for the native enzyme. In addition, the time course of the formation of the various fragments was followed by measuring enzymatic activity and performing gel electrophoretic analysis of the protein mixture at defined time intervals during proteolysis. On the basis of the results of these studies, a reaction scheme describing the succession of events during proteolysis is given.