John S. Easterby

Coupled enzyme assays: A general expression for the transient

Abstract The rate of formation of product in a coupled enzyme reaction is subject to a transient phase prior to the attainment of the steady-state. General expressions are presented to describe the transient in a multi-enzyme sequence in which the initial enzyme is rate-limiting. This reveals that the initial enzyme does not contribute to the transient but does determine the steady-state velocity. Each coupling enzyme has a characteristic transient time given by the ratio of its Michaelis constant to its maximum velocity. The transient for the complete sequence is a simple sum of the individual enzyme transients. In metabolic sequences for which the initial enzyme is rate-limiting the lag phase will be controlled by the enzymes of the sequence with greatest transient times and not by the initial rate-limiting enzyme.

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.

[2] Hexokinase type II from rat skeletal muscle

Publisher Summary This chapter describes an affinity elution procedure that is adopted in the purification of Hexokinase type II, which is the principal isozyme of rat skeletal muscle. The procedure of purification is highly specific, but, unlike affinity chromatography, does not require the synthesis of a matrix containing immobilized ligand. Hexokinase type II is extremely unstable and is rapidly inactivated in the absence of hexose or thiol, which makes purification difficult. The glucose 6-phosphate produced in the hexokinase reaction is coupled to glucose-6-phosphate dehydrogenase. The reaction is followed by measuring the increase in absorbance at 340 nm because of NADPH formation. The molecular weight of hexokinase determined by sedimentation equilibrium is 100,000 and the isoelectric point is measured as pH 5.7 by analytical isoelectric focusing.

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.

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.

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 bakers 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.

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.

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.

Temporal Analysis of the Transition between Steady States

Most Studies of metabolic control have concentrated on defining pathway flux and its determinants. In particular the fine-control of enzyme activity and the homeostatic maintenance of steady states has been a focus of attention. In the cell many changes are more dramatic and involve large changes of flux. Much less attention has been given to the equally important topic of the time-scale on which such changes, and metabolic processes generally, operate. A full description of regulation of a system must include such temporal analysis. It is insufficient to ask of a pathway, cell or organism “How fast does it go?”, it is also necessary to ask “How long does it take to get there?”. This temporal responsiveness sets the time-scale on which metabolism operates and is best described by the pathway transition or transient time, τ. The reluctance to study pathway dynamics probably arises partly from a mistaken belief that real-time measurements or a detailed knowledge of pathway kinetics is necessary. Either way the experimentation would be difficult.

Purification of heart hexokinase by dye-ligand chromatography

Abstract Heart hexokinase (EC 2.7.1.1) has been purified by a procedure that uses affinity elution from a column of red triazine dye, H-8BN, immobilized to Sepharose 6B. Homogeneous protein (9 mg) is obtained from 500 g heart in 40% yield. The mechanism of affinity elution from the dye is discussed.