Metabolic channeling versus free diffusion: transition-time analysis.

Metabolic channeling is the term used to describe the restricted flow of substrates and products in multienzyme systems. It has been argued for some time that free diffusion is sufficiently rapid to obviate the need for channeling and, furthermore, that it is also fast enough to prevent competing side reactions from interfering with the metabolic flow. In this article we argue that a thorough consideration of the temporal behavior of metabolite pools suggests that channeling is important in many cases.

Comparison of type I hexokinases from pig heart and kinetic evaluation of the effects of inhibitors.

Type I hexokinase (ATP:D-hexose 6-phospotransferase, EC 2.7.1.1) of porcine heart exists in two chromatographically distinct forms. These do not differ significantly in size, electrophoretic mobility at pH 8.6 or kinetic properties. Both forms obey a sequential mechanism and are potently inhibited by glucose 6-phosphate. In contrast to observations of type I hexokinase from brain, inhibition by glucose 6-phosphate is not relieved by inorganic phosphate. Under most conditions, low concentrations of phosphate (less than 10 mM) have little effect on the kinetic behaviour of the enzyme but at higher concentrations this ligand is an inhibitor. Mannose 6-phosphate inhibits in a manner analogous to glucose 6-phosphate but the Ki is much greater. In view of the similarity of the kinetic parameters governing phosphorylation of mannose and glucose, this difference in affinity for the inhibitor site is seen as consistent with the existence of a separate regulatory site on the enzyme. MgADP inhibits hexokinase but behaves as a normal product inhibitor and inhibition is competitive with respect to MgATP and non-competitive with respect to glucose.

Purification and molecular properties of bovine heart pyruvate kinase.

A rapid method is presented for the purification of pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from bovine heart. The enzyme obtained is homogeneous by criteria of sodium dodecyl sulphate polyacrylamide electrophoresis and ultracentrifugation and has a specific activity of 260 units/mg. It is a tetramer of 220 000 daltons and S020,w = 10.0 S and possesses no free amino-terminal residue. The amino acid composition is similar to that of the M1 isozyme of rabbit and bovine skeletal muscle. The enzyme is subject to polymerisation to a hexamer of the basic tetramer. The polymeric species has a molecular weight of 1320 000, is promoted at low ionic strength and is undetectable at ionic strength greater than 0.2 by either sedimentation equilibrium or sedimentation velocity measurements. The polymerisation is independent of temperature in the range 5--20degrees C implying that charge interactions rather than apolar interactions are responsible for the process.

Isolation of multiple dimeric forms of phosphoribulokinase from an alga and a higher plant.

Dimeric phosphoribulokinase from either spinach (Spinacia oleracea) leaf or from the green alga, Scenedesmus obliquus can be separated into three distinct forms by hydrophobic interaction chromatography. Variation of the redox conditions prior to and during chromatography resulted in specific forms of phosphoribulokinase being eluted. It is suggested that three dimeric forms of phosphoribulokinase differ in the extent of disulfide bond formation between Cys-16 and Cys-55 in each of the two subunits. Phosphoribulokinase-3, isolated under the most oxidising conditions and exhibiting unusual kinetics, has properties consistent with those expected of an oxidised form of the enzyme in which Cys-16 and Cys-55 are completely oxidised to form a disulfide bond in each subunit. Phosphoribulokinase-1 is the completely reduced form predominating following incubation of extracts with dithiothreitol. Phosphoribulokinase-2, the intermediate species in which only one subunit possesses the disulfide, predominates only when extracts, previously reduced by high concentrations of 2-mercaptoethanol, are allowed to stand overnight in the presence of air prior to chromatography.

Algal glyceraldehyde-3-phosphate dehydrogenases. Conversion of the NADH-linked enzyme of Scenedesmus obliquus into a form which preferentially uses NADPH as coenzyme.

Scenedesmus obliquus contains two glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.-) one of which uses NADH as its preferred coenzyme (D-enzyme) and the other NADPH (T-enzyme). On incubation of the D-enzyme with cysteine and a 1,3-diphosphoglycerate-generating system the specific activity with NADH as coenzyme decreased whilst that with NADPH increased by a factor of 10. The components of the generating system had no effect on the D-enzyme individually and it is concluded that 1,3-diphosphoglycerate was probably responsible for the change in nucleotide specificity. The coenzyme specificity of the T-enzyme was not affected by such treatment. A similar type of activation occurred to a lesser extent on incubation of the D-enzyme with 2,3-diphosphoglycerate. The NADPH-dependent activity of the D-enzyme could also be promoted by incubation with NADPH. However, in this case the activation was less than that seen with either 1,3- or 2,3-diphosphoglycerate. The change in coenzyme specificity of the D-enzyme occurred in parallel with changes in sedimentation behaviour. Initially, a single boundary of S20,w equals 14.5 S was present, but on conversion to NADPH-dependent activity by incubation with the 1,3-diphosphoglycerate-generating system, new boundaries of 7.5 S and 5.5 S appeared. The first of these corresponds in sedimentation coefficient to the native T-enzyme. On removal of 1,3-diphosphoglycerate the 7.5 S boundary disappeared accompanied by an increase in that of 14.5 S, whilst the 5.5 S boundary persisted. These changes are consistent with the reversible conversion of the D-enzyme into a form similar to the native T-enzyme in response to cysteine and 1,3-diphosphoglycerate. These effects may be explained if acylation of the active site of the D-enzyme by 1,3-diphosphoglycerate results in displacement of the bound nucleotide, thus promoting nucleotide exchange. These findings are consistent with the kinetic mechanism established for other glyceraldehyde-3-phosphate dehydrogenases. Similar activation was seen in extracts of other species of the Chlorophyta but not in other photosynthetic organisms. The significance of this type of activation of enzyme activity to the metabolism of these species of algae is discussed.

Glyceraldehyde-3-phosphate dehydrogenase of Scenedesmus obliquus. Effects of dithiothreitol and nucleotide on coenzyme specificity.

NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.--) of the photosynthetic alga Scenedesmus obliquus is converted to an NADPH specific form by incubation with dithiothreitol. The change in nucleotide specificity is accompanied by a reduction in the molecular weight of the enzyme from 550 000 to 140 000. Prolonged incubation with dithiothreitol results in the further dissociation of the enzyme to an inactive 70 000 dalton species. The 140 000 dalton, NADPH-specific enzyme is stabilized against dissociation and inactivation by the presence of NAD(H) or NADP(H). Optimum stimulation of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase activity is achieved on incubation of the NADH-specific enzyme with dithiothreitol and NADPH, or dithiothreitol and a 1,3-diphosphoglycerate generating system. The relevance of these observations to in vivo light-induced changes in the nucleotide specificity of the enzyme is discussed.

The analysis of metabolite channelling in multienzyme complexes and multifunctional proteins.

Multienzyme complexes and multifunctional proteins may confer a kinetic advantage by channelling reaction intermediates between consecutive enzymes and reducing the transient time for the establishment of steady states. A general means for quantitatively assessing the contribution of channelling to the reduction of pool size and transient time is presented. Restrictions to the kinetic advantage are identified, and it is shown that no channelling advantage is obtained at high enzyme concentration or for enzymes which exhibit rapid-equilibrium kinetic behaviour.

The kinetics of consecutive enzyme reactions. The design of coupled assays and the temporal response of pathways.

A regime is proposed for the design of coupled enzyme assays in which auxiliary enzymes are added at concentrations proportional to their Km values. Under these conditions it is possible to calculate the complete time course of the assay including the time required for the system to approach its steady state. The consequence of increasing the number of coupling enzymes is shown to be a considerable decrease in time required to reach the steady state provided that the overall transient time remains the same. The method is extended to the general consideration of pathways and shows that pathways of the same length exhibit identical temporal responses provided that the units of concentration and time used are based on the steady-state concentration of intermediates and the transient time respectively. An unexpected finding is that increasing the number of intermediates in a pathway can decrease the time required to enter a steady state.

A generalized theory of the transition time for sequential enzyme reactions.

In a sequence of coupled enzyme reactions the steady-state production of product is preceded by a lag period or transition time during which the intermediates of the sequence are accumulating. Provided that a steady state is eventually reached, the magnitude of this lag may be calculated, even when the differentiation equations describing the process have no analytical solution. The calculation may be made for simple systems in which the enzymes obey Michaelis-Menten kinetics or for more complex pathways in which intermediates act as modifiers of the enzymes. The transition time associated with each intermediate in the sequence is given by the ratio of the appropriate steady-state intermediate concentration to the steady-state flux. The theory is also applicable to the transition between steady states produced by flux changes. Application of the theory to coupled enzyme assays allows a definition of the minimum requirements for successful operation of the assay. The theory can be extended to deal with sequences in which the enzyme concentration exceeds substrate concentration.

Integration of temporal analysis and control analysis of metabolic systems.

A theory is developed that integrates approaches to the analysis of pathway transient response and metabolic control analysis. A Temporal Control Coefficient is defined that is a measure of the system's transient response to modulation of enzyme activity or concentration. The approach allows for the analysis of the establishment of a steady state from rest, of the system's 'agility' of response to minor perturbations of a pre-existing steady state and of the macroscopic transition between steady states. In the last-mentioned case it is shown that, like the transient time itself, the control of transient response retains the property of independence from the mechanism of the transition. In consequence, the Temporal Control Coefficient can be defined in terms of the control properties of the initial and final states alone without reference to the mechanism of transition. A summation property is shown to apply to the Temporal Control Coefficients in each case. Connectivity relationships between elasticities and Temporal Control Coefficients are also established.

The effect of feedback on pathway transient response.

The effect of variation of the rate of input of material on the transient behaviour of metabolic pathways is examined. This reveals the existence of three transient times which make up the overall pathway transient. Two of these have been described previously and represent the times required for the accumulation of the free intermediate pool and the pool of enzyme-bound intermediate. They are state functions and as such are independent of the way in which the steady state was reached. The third is attributable to the variation in the rate of input of material to the pathway. It is dependent on three further factors. These are (a) the time required for the initial enzyme to reach its own steady state, (b) substrate depletion and (c) feedback. The description of the transient is: (Formula: see text) where V0 represents the rate of input and Vss represents the steady-state flux. The transient time associated with the transition between steady-states is shown to be a simple function of the transients for the establishment of each steady state from rest and may be expressed as: tau = tau b-Va/Vb . tau a where Va and Vb refer to the fluxes in the two steady states and tau a and tau b represent the transient times for the establishment of each of the steady-states from rest. The total pathway transient may now be completely defined as: (formula: see text) where summation over all intermediates, I, is implied. The significance of this to the analysis of pathway behaviour is discussed with more general examples of pathway transient analysis.

The fusion of control analysis and temporal analysis of metabolic systems.

Metabolic control analysis and the study of the transient response of metabolic systems had coincident births in 1973. They developed along parallel lines until in 1989/90 their complete fusion occurred. It was evident that the control of the transient response of metabolism could be described in terms of general control properties, such as the flux and concentration control coefficients and elasticities. Consequently, it is possible to define temporal control coefficients which relate to the lifetimes of individual metabolite pools or to the total system temporal response. These control coefficients are readily expressed in terms of the flux and concentration control coefficients. Therefore, to analyse the control of metabolism is also to analyse its temporal response.

Temporal analysis of metabolic systems and its application to metabolite channelling.

When a metabolic system undergoes a transition between steady states, the lag or transition time of the system is determined by the aggregated lifetimes of the metabolite pools. This allows the transition time, and hence the temporal responsiveness of the system, to be estimated from a knowledge of the starting and finishing steady states and obviates the need for dynamic measurements. The analysis of temporal response in metabolic systems may be integrated with the general field of metabolic control analysis by the definition of a temporal control coefficient (Cei tau) in terms of flux and concentration control coefficients. The temporal control coefficient exhibits summation and other properties analogous to the flux and concentration control coefficients. For systems in which static metabolite channels exist, the major kinetic advantage of channelling is a reduction in pool sizes and, as a result, a more rapid system response reflected in a reduced transition time. The extent of the channelling advantage may therefore be assessed from a knowledge of the system transition time. This reveals that no channelling advantage is achieved at high enzyme concentrations (i.e., comparable to Km) or, in the case of 'leaky' channels, where rapid equilibrium kinetic mechanisms obtain. In the case of a perfect channel with no leakage and direct transfer of metabolite between adjacent enzyme active sites, the transition time is minimized and equal to the lifetime of the enzyme-substrate complex.

Heart hexokinase: quaternary structure changes accompanying the binding of regulatory molecules.

Heart hexokinase monomer has a molecular weight of 97000 and so20,w 5.2 S. It exists in equilibrium with dimer of 194000 molecular weight and so20,w 8.1 S. The proportions of monomer and dimer presence of added ligands are 91% and 9% respectively. The existence of these forms may be demonstrated by separation on electrophoresis or chromatography. In the presence of the regulatory molecule glucose 6-phosphate, the dimer form of the enzyme is favoured. The glucose 6-phosphate mediated dimerisation is abolished in the presence of phosphate or ATP-Mg and less effectively by free ATP. Glucose has no effect on the manomer-dimer equilibrium. On prolonged storage of hexokinase in glucose 6-phosphate polymers are also formed and polymerisation is further enhanced by removal of the ligand.

The purification of yeast glucose 6-phosphate dehydrogenase by dye-ligand chromatography.

Glucose 6-phosphate dehydrogenase (EC 1.1.1.39) has been purified to homogeneity from baker's yeast by a simple procedure involving affinity elution from a column of red triazine dye, H-8BN, immobilized to Sepharose 6B. Eight milligrams of homogeneous protein is obtained in 53% yield from 200 g of dried yeast. This represents the first published purification of the enzyme from Saccharomyces Cerevisiae.

Wheatgerm hexokinase (LII): fluorimetric measurement of the binding of substrates and products.

The change in intrinsic fluorescence observed when wheatgerm hexokinase combines with its substrates or products has been investigated. The dissociation constants for the enzyme - ligand complexes have been evaluated and found to be equal to their respective Michaelis constants, and confirm that fructose is the preferred hexose substrate. Both hexoses and nucleotides can bind independently to the enzyme and the data are consistent with previous proposals that conformation changes in the enzyme may accompany the random binding of substrates.

Turnover of glycogen phosphorylase in the pectoralis muscle of broiler and layer chickens.

Glycogen phosphorylase is a major sarcoplasmic protein in chicken pectoralis muscle, constituting approx. 4% of the total protein complement. In slow-growing layer chicks phosphorylase accumulated in parallel with muscle accretion, but in fast-growing broiler chicks the concentration of phosphorylase in the muscle increased (from 5 to 8 mg/g wet wt.) with time. In a 5-week period, the total amount of phosphorylase in the pectoralis muscles increased 18-fold in broiler chicks (from approx. 75 to 1400 mg total), but only 3-fold (from approx. 100 to 270 mg total) in layers. Pyridoxal phosphate, the cofactor of the enzyme glycogen phosphorylase, was used as a specific label to measure the rate of degradation of the enzyme in the pectoralis muscle of growing broiler and layer chickens in vivo. In young animals, the fractional rate of phosphorylase synthesis was similar in broiler and layer chickens (approx. 15%/day), but the rate of degradation in layers (5%/day) was 5-fold higher than in broilers (1%/day). As the animals aged, the rate of synthesis decreased, but more so in layers than in broilers. The rate of degradation of phosphorylase also decreased in layers, but in broilers it remained at the low level seen in young animals. The dramatically higher rate of phosphorylase accretion in the pectoralis muscles of the broilers is therefore achieved by an initial lower rate of degradation combined with a sustained difference between rates of synthesis and degradation.

Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

A homogeneous multimeric protein isolated from the green alga, Scenedesmus obliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560,000 and was of apparent subunit composition 8G6R. R represents a subunit of Mr 42,000 conferring phosphoribulokinase activity and G a subunit of 39,000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42,000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of Mr-39,000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. The multimeric enzyme had a S20,W of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4G) and active phosphoribulokinase (2R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography. Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase. Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.