Competitive inhibition of lipolytic enzymes. I. A kinetic model applicable to water-insoluble competitive inhibitors.

It is now becoming clear from the abundant lipolytic enzyme literature that any meaningful interpretation of inhibition data has to take into account the kinetics of enzyme action at the lipid/water interface. We attempt in the present paper to provide a kinetic model applicable to water-insoluble competitive inhibitors, in order to quantitatively compare the results obtained at several laboratories. We derived kinetic equations applicable to the pre-steady state as well as steady state. By measuring the inhibitory power, as described in the present paper, it is possible to obtain a normalized estimation of the relative efficiency of various potential inhibitors. Furthermore, with the kinetic treatment developed here, it is possible to make quantitative comparisons with the same inhibitor placed under various physico-chemical situations, i.e., micellar or monolayer states.

Equilibrium binding of thioredoxin fB to chloroplastic fructose bisphosphatase. Evidence for a thioredoxin site distinct from the active site.

Thioredoxin fB, the protein activator of chloroplastic fructose 1,6-bisphosphatase, strongly binds its target enzyme with a stoichiometry of one protein dimer per enzyme tetramer. The thioredoxin binding site is distinct from the active site and the dissociation constant of the protein-enzyme complex has the extremely small value of 769 nM at pH 7.5. This interaction involves both ionic and hydrophobic contributions and is enhanced by a pH increase from 7 to 8. These results suggest that the above molecular properties may be involved in the light activation of chloroplastic fructose bisphosphatase.

Enzymes as biosensors. 2. Hysteretic response of chloroplastic fructose-1,6-bisphosphatase to fructose 2,6-bisphosphate.

Oxidized chloroplastic fructose-bisphosphatase is almost totally inactive at pH 7.5, that is under pH conditions that prevail in the chloroplast stroma. When preincubated for different time periods with fructose 2,6-bisphosphate and assayed in the absence of this ligand, it displays an activity which is directly related to the duration of the preincubation phase. This implies that fructose 2,6-bisphosphate induces enzyme conformers that appear in sequence and may be competent for catalytic activity. Upon desorption of fructose 2,6-bisphosphate the enzyme may retain its active conformation for a time period whose duration depends on magnesium concentration. It thus appears that reduction of the enzyme is not an obligatory prerequisite for its activity. Fructose 2,6-bisphosphate behaves as a competitive inhibitor of the reduced, active enzyme, with respect to the real substrate. When assayed with the oxidized enzyme, however, it behaves as an activator. Moreover the apparent steady-state rate that may be measured experimentally depends on both fructose 2,6-bisphosphate concentration and the direction of a concentration change. The reaction velocity experimentally measured is thus a meta-steady-state rate and depends on the initial conditions of the system. The fructose-bisphosphatase system thus displays, with respect to fructose 2,6-bisphosphate, a hysteresis loop and may then sense whether the concentration of that ligand is increased or decreased. A model has been proposed which allows one to explain these results. This model is based on the view that the substrate and fructose 2,6-bisphosphate compete for the same site of the enzyme and that this latter ligand stabilizes a conformation competent for enzyme activity. After the ligand has been chased away, the enzyme retains the active conformation for a while and slowly relapses to the initial inactive conformation. The time-scale of this slow relaxation overlaps that of the steady state of product appearance and this generates meta-steady-state kinetics, which is dependent on the initial state and therefore on the history of the system.

Potential memory and hysteretic effects in transcription.

Kinetic results of RNA-chain elongation catalysed by wheat-germ RNA polymerase II are analysed according to the concept that DNA-dependent conformational transitions of the transcription complex intervene during transcription. A model is presented, involving participation of several forms of the transcription complex with different catalytic properties, generated by the sequence and/or conformation of the DNA template and/or the experimental conditions. The available experimental data suggest that these forms are interconvertible. Examples in which hysteretic transitions might occur are outlined, such as termination of transcription and transition from abortive to productive elongation in the first steps of RNA synthesis. The slow catalytic adaptation of the transcription complex to the template sequence might be a more general phenomenon for enzyme systems acting on polynucleotide templates, in view of the recent proposal that enzyme memory effects may also have some importance in DNA replication and messenger RNA (mRNA) translation.

Kinetic implications of the occurrence of several relaxations in the conformational transition of mnemonical enzymes.

If the conformational transition involved in enzyme memory occurs in several elementary steps, the time constant of the overall 'slow' relaxation is mostly determined by the individual values of the rate constants pertaining to the overall transconformation. The extent of kinetic co-operativity of the enzyme reaction, however, is mostly controlled by the degree of reversibility of the elementary steps of the conformational transition. There is then no simple relation between the time scale of the 'slow' transition and the extent of kinetic co-operativity of the enzyme reaction. A slow transition of about 10(-3) s-1 is therefore perfectly compatible with a strong positive or negative co-operativity and in particular with the negative co-operativity observed with wheat germ hexokinase LI. The relationship that has been established recently [Pettersson, G. (1986) Eur. J. Biochem. 154, 167-170] between the 'slow' enzyme relaxation and the extent of kinetic co-operativity holds only in the specific case where the transconformation occurs in one step. Owing to the possible occurrence of a multistep conformation change, the lack of this relationship means nothing as to the validity, or the invalidity, of the concept of mnemonical transition. More informative than the time scale of the 'slow' transition is its dependence with respect to glucose and glucose 6-phosphate, which both react with the enzyme. The effect of reaction products on the modulation of kinetic co-operativity is also of cardinal importance in the diagnosis of enzyme memory. Since an alternative model has been recently proposed by Pettersson (cited above) to explain the mechanistic origin of kinetic co-operativity of monomeric enzymes, the effect of products on the kinetic co-operativity predicted by this alternative model has been studied theoretically, in order to determine whether it is consistent with the experimental results obtained with wheat germ hexokinase LI. This analysis shows that the predictions of this model are in total disagreement with both the predictions of the mnemonical model and the experimental results obtained with wheat germ hexokinase LI, as well as with other enzymes. This alternative model cannot therefore be considered as a sensible explanation of the mechanistic origin of co-operativity of monomeric enzymes. It is therefore concluded that the mnemonical model which rests on numerous experimental results, obtained by different research groups, on different enzymes is the simplest and most likely explanation of the kinetic subtleties displayed by some monomeric enzymes, and in particular wheat germ hexokinase LI.

Kinetics of the conformational transition of the spinach chloroplast fructose-1,6-bisphosphatase induced by fructose 2,6-bisphosphate.

The activation of oxidized chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate and magnesium previously described at pH 7.5 [Soulié et al. (1988) Eur. J. Biochem. 176, 111-117] has now been studied at pH 8, the pH which prevails under light conditions in the chloroplast stroma. The process obeys a hysteretic mechanism but the rate of activation is considerably increased with half-times down to 50 s and the apparent dissociation constant of fructose 2,6-bisphosphate from the enzyme is lowered from 1 mM at pH 7.5 to 3.3 microM at pH 8. The process is strictly metal-dependent with a half-saturation concentration of 2.54 mM for magnesium. The conformational transition postulated in our hysteretic model has been investigated through both the spectrophometric and chemical modification approaches. The activation of the enzyme by fructose 2,6-bisphosphate in the presence of magnesium results in a slow modification of the ultraviolet absorption spectrum of the enzyme with an overall increase of 3% at 290 nm. The same treatment leads to the protection of two free sulfhydryls and an increased reactivity of one sulfhydryl group/enzyme monomer to modification by 5,5'-dithiobis(2-nitrobenzoic acid). The titration of the exposed cysteinyl residue prevents the relaxation of enzyme species induced by fructose 2,6-bisphosphate to the native form. The activation of chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate is discussed both with respect to the understanding of the overall regulation properties of the enzyme and to a possible physiological significance of this process.

Kinetic co-operativity of wheat-germ RNA polymerase II with adenosine 5'-[beta gamma-imido]triphosphate as substrate.

A kinetic study of productive RNA chain elongation indicates that adenosine 5'-[beta gamma-imido]triphosphate can serve as substrate in reactions catalysed by purified wheat-germ RNA polymerase II on a poly[d(A-T)] template. However, in contrast with the results obtained with the natural substrate ATP, the double-reciprocal plots, 1/velocity versus 1/[nucleotide], are not linear but characteristic of apparent negative co-operativity. The extent of the kinetic co-operativity is modified when the reactions are conducted in the additional presence of fixed amounts of an alternative substrate such as ATP or inhibitors such as dATP or cordycepin triphosphate. Analogous results are obtained whether the reactions are carried out in the presence of Mg2+ or Mn2+ as the metal ion cofactor. However, the data show that with Mn2+ the RNA polymerase is less specific in substrate recognition than with Mg2+. Tentative kinetic models are proposed to account for the rate measurements.

Transient kinetic studies of fatty acid synthetase. A kinetic self-editing mechanism for the loading of acetyl and malonyl residues and the role of coenzyme A.

A kinetic self-editing mechanism for correcting errors in the loading of thioester substrates is described for the animal fatty acid synthetase reaction. In the catalyzed reaction, these substrates load competitively on a common phosphopantetheine site, and during each of the eight loading steps the enzyme sites are partitioned between competent and incompetent substrate molecules. The incompetently bound substrate is removed by CoA through reversal of the loading reaction and partitioning again occurs. The loading-unloading cycle is repeated until competent enzyme complex is formed and the reaction proceeds. Furthermore, at each step the loading of a malonyl residue is competitively favored as is the unloading of enzyme-bound acetyl groups. This mechanism is entirely consistent with the recently postulated role (Stern, A., Sedgwick, B., and Smith, S. J. Biol. Chem. (1982) 257, 799-803) of CoA as a co-substrate. Supporting evidence is obtained by monitoring the progress curves of NADPH oxidation by chicken liver fatty acid synthetase in the stopped flow apparatus. At noninhibiting acetyl-CoA, the reaction shows an initial lag period as the result of preferential formation of malonyl-enzyme and time-dependent recycling of the loading step to obtain competent acetyl-enzyme. At a malonyl-CoA/acetyl-CoA ratio of 2:1, the induction time of the reaction is 1.02 +/- 0.05 s at 6 degrees C. It decreases with increasing acetyl-CoA concentration or preincubation of the enzyme with acetyl-CoA which promotes acetyl-enzyme formation but is slightly increased upon preincubation with malonyl-CoA. Increasing acetyl-CoA causes a parallel decrease in steady state cycle time (i.e. the average time required to complete a single malonyl-CoA condensation cycle), suggesting that the latter is limited by the lag period. At inhibitory acetyl-CoA, the steady state cycle time is lengthened due to acetyl-enzyme formation at malonyl-CoA loading steps and to the recycling necessary to obtain competent malonyl-enzyme. A requirement of CoA for the first condensation cycle is unequivocally demonstrated in conventional spectrophometric assays and stopped flow experiments by using phosphotransacetylase and acetyl phosphate as a CoA trap. This requirement at each loading step is normally met by CoA generated through initial loading. At noninhibitory acetyl-CoA, added CoA inhibits the reaction and slightly increases the lag.(ABSTRACT TRUNCATED AT 400 WORDS)

On the activation of fructose-1,6-bisphosphatase of spinach chloroplasts and the regulation of the Calvin cycle.

Activation by light of spinach fructose-1,6-bisphosphatase is mimicked by dithiothreitol. This process of activation by dithiothreitol implies the specific reduction of two disulfide bridges and a conformation change of the enzyme that makes eight sulfhydryl groups available. The activated enzyme has an apparent allosteric kinetic behavior with respect to both fructose 1,6-bisphosphate and magnesium.

Substrate-binding isotherms of spinach chloroplastic fructose-1,6-bisphosphatase and the photoregulation of the Calvin cycle.

Fluorescence titration experiments of chloroplastic fructose-1,6-bisphosphatase by fructose bisphosphate and magnesium have been effected using the inactive dimeric, the inactive tetrameric and the active tetrameric enzyme forms. Magnesium binding to the inactive dimeric enzyme exhibits a positive cooperativity whereas fructose 1,6-bisphosphate exhibits no cooperativity at all. The binding of either magnesium or fructose bisphosphate to the inactive oxidized tetramer exhibits a succession of negative and positive cooperativities (mixed cooperativity). Upon reduction of the inactive tetramer by dithiothreitol and activation, the ligand binding properties of the enzyme are changed. Magnesium and fructose bisphosphate are bound to the active enzyme with a positive cooperativity. Non-linear least-square fitting allows one to estimate thea binding constants, and therefore the free energy of binding of either magnesium or fructose bisphosphate to the various forms of the enzyme. Whatever the state of fructose-1,6-bisphosphatase, one ligand is bound per subunit. The affinity constants of magnesium for the active or inactive enzyme forms are much greater than those of fructose bisphosphate. This suggests that the metal ion gives to the enzyme the right conformation to bind the sugar phosphate.

Kinetics of dissociation of reduced nicotinamide adenine dinucleotide phosphate from its complexes with malic enzyme in relation to substrate inhibition and half-of-the-sites reactivity.

Malic enzyme of pigeon liver binds NADPH at four equivalent enzyme sites and binds Mn2+ and malate each at two sets of "tight" and "weak" sites with negative cooperativity [Pry, T. A., & Hsu, R. Y. (1980) Biochemistry 19, 951-962]. Stopped-flow studies on the displacement of NADPH from the malate-enzyme complexes E4-NADPH4, E4-Mn2(2+)-NADPH4, E4-Mn2(2+)-NADPH4-dimalate, and E4-Mn2(2+)-NADPH4-tetramalate by large excess NADP+ or its analogue phosphoadenosine(2')diphospho(5')ribose show that NADPH dissociates from the binary complex rapidly with a first-order rate constant of 427 s-1. Dissociation from the ternary E4-Mn2(2+)-NADPH4 complex containing two tightly bound Mn2+ ions can be described by a single first-order process with a rate constant of 135 s-1, or more satisfactorily by two simultaneous first-order processes attributable to the reactions of Mn2+-deficient (k congruent to 427 s-1) and Mn2+-liganded (k = 96 s-1) subunits. The latter equals twice the maximum steady-state turnover rate of 53.2 + 3.0 s-1 assigned to dissociation of the reduced nucleotide from transient E-Mn2+-NADPH, and this 2:1 ratio strongly supports our proposed "half-of-the-sites" model [Hsu, R. Y., & Pry, T. A. (1980) Biochemistry 19, 962-968]. Dissociation from the E4-Mn2(2+)-NADPH4-dimalate complex (k = 100 s-1) follows only the slower process, suggesting that occupancy of malate at two sites tightens enzyme-bound NADPH on the adjacent sites. Binding of malate at two additional weak sites yields E4-Mn2(2+)-NADPH4-tetramalate and a NADPH dissociation rate constant of 2.69 s-1. The 97% decrease in NADPH dissociation parallels the observed 93% maximal inhibition by malate and is the cause of substrate inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes.

In vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to determine whether or not the newly synthesized aminopeptidase N transits through the basolateral membrane before it reaches the apical brush border, its final localization. The kinetics of the arrival of the newly synthesized enzyme in the Golgi complex, basolateral and brush border membrane fractions strongly suggest that on leaving the Golgi aminopeptidase N is transiently integrated into the basolateral domain before reaching the brush border.

Kinetics of the modulation of chloroplastic fructose-1,6-bisphosphatase activity by thioredoxin fb.

The inactivation of reduced chloroplast fructose-bisphosphatase by oxidized thioredoxin fb has been studied during the enzyme reaction along the principle of Tian and Tsou [Biochemistry (1982) 21, 1028-1032]. A minimum model for this process is presented and its kinetic and equilibrium parameters have been determined. Thioredoxin fb binding to the enzyme is fast relative to catalysis and product desorption. Under quasi-equilibrium conditions oxidized thioredoxin is a non-competitive inhibitor of the enzyme reaction and must bind to a regulatory 'thioredoxin site'. The slow deactivation is thermodynamically favoured, and as expected from binding data, slowed down by the presence of substrate, fructose bisphosphate. The desorption of thioredoxin fb from the enzyme is extremely slow and this small protein may be regarded as a 'regulatory' subunit of fructose-bisphosphatase.

Abortive intermediates in transcription by wheat-germ RNA polymerase II. Dynamic aspects of enzyme/template interactions in selection of the enzyme synthetic mode.

At constant enzyme concentration and with the full set of nucleotide substrates dictated by template sequence, the chain-length distribution of polymeric product varies with template concentration in reactions catalysed by wheat-germ RNA polymerase II. Under the same conditions, but in the presence of a single ribonucleoside triphosphate, the rate of condensation of the triphosphate substrate to a dinucleotide primer also exhibits a complex dependence with the template concentration. This effect is observed using poly[d(A-T)] as a template. For both reactions there are two extreme types of behaviour in each of which transcription appears to involve a single enzyme synthetic mode, characterized by either a high (at low template concentration) or a low (at high template concentration) probability of releasing the transcripts. A strong correlation is found between these two pathways, such that conditions favouring the abortive release of trinucleotide products in the single-step addition reaction are associated with the synthesis of short-length RNA species in productive elongation, and reciprocally. A model previously developed by Papanicolaou, Lecomte & Ninio [(1986) J. Mol. Biol. 189, 435-448] to account for the kinetics of polymerization/excision ratios with Escherichia coli DNA polymerase I, and by Job, Soulié, Job & Shire [(1988) J. Theor. Biol. 134, 273-289] for kinetics of RNA-chain elongation by wheat-germ RNA polymerase II provides an explanation for the observed behaviour with the plant transcriptase. The basic requirement of this model is a slow equilibrium between two states of the polymerization complex with distinct probabilities of releasing the product. In the presence of Mn2+, and under conditions allowing the synthesis of poly[r(A-U)], one of these states is involved in the formation of oligonucleotides shorter than 15 bases, whereas the other catalyses the polymerization of chains longer than 40 bases.