Interconvertible Enzyme Cascade Research Articles

A kinetic re-interpretation of the regulation of rabbit skeletal-muscle phosphorylase kinase activity by Ca2+ and phosphorylation.

The regulation of phosphorylase kinase has been proposed to occur physiologically under conditions of zero-order ultrasensitivity [Meinke & Edstrom (1991) J. Biol. Chem. 266, 2259-2266]. This is also one of the conditions that recent theoretical approaches have indicated to be essential in order for an interconvertible enzyme cascade to generate a sensitive response to an effector [Cardenas & Cornish-Bowden (1989) Biochem. J. 257, 339-345]. In contrast, all published kinetic data to date have strongly suggested that activation of phosphorylase kinase by Ca2+ or phosphorylation is attributable solely to a change in affinity for phosphorylase, with no effect on the Vmax. of the reaction. In this study an attempt is made to resolve this conflict. Findings suggest that changes in Vmax. can fully account for the activation of phosphorylase kinase by the physiological mechanisms of cyclic AMP-dependent phosphorylation and increase in Ca2+ concentration.

Response coefficients of interconvertible enzyme cascades towards effectors that act on one or both modifier enzymes.

Explicit expressions have been derived for the response coefficients for the effect of activator and inhibitor concentrations on the fraction in the active state of the target enzyme of a monocyclic interconvertible enzyme cascade. These allow one to assess the adequacy of such a cascade for producing a highly sensitive response to an effector. Numerical studies indicate that this type of system can readily generate response coefficients of about seven, even without requiring both modification reactions to be modulated simultaneously, and without requiring all of the parameters that characterize the system to have their optimum values. Thus, a monocyclic cascade can constitute a highly effective on/off switching device in a linear pathway.

Characteristics necessary for an interconvertible enzyme cascade to generate a highly sensitive response to an effector.

A monocyclic interconvertible enzyme cascade, in which active and inactive states of an enzyme are interconverted by two opposing enzyme-catalysed reactions, does not necessarily produce a greater degree of sensitivity to an effector than one could expect from direct interaction between effector and target reaction. On the contrary, a cascade in which an effector acts on one of the enzymes catalysing the interconversion reactions by altering the apparent value of its specificity constant will always generate a less sensitive response than direct interaction would give. Nonetheless, even if both interconversion reactions obey Michaelis-Menten kinetics with the ordinary types of inhibition and activation, one can easily generate an enormous sensitivity in which a 0.5% change in concentration can increase the proportion of target enzyme in the active state from 10% to 90%: this corresponds approximately to a Hill coefficient of 800. To maximize the sensitivity, the following conditions must be satisfied: (1) both modifier enzymes must act under conditions of near saturation; (2) the effector must act on both of them in opposite directions; (3) it must alter the apparent values of their catalytic constants; (4) the enzyme subject to inhibition by the effector must respond at much lower effector concentrations than the enzyme subject to activation. As the last of these conditions appears to be counter-intuitive, it suggests that feeble activation of modifier enzymes in real systems may have passed unnoticed, or been dismissed as physiologically insignificant, although in reality crucial to the effective response of the system.

Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Escherichia coli. Studies of monocyclic and bicyclic interconvertible enzyme cascades, in situ.

Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Escherichia coli. Studies of monocyclic and bicyclic interconvertible enzyme cascades, in situ.

12] Covalently interconvertible enzyme cascade systems

Publisher Summary This chapter reviews that the cyclic cascade systems are fundamentally different from the irreversible, unidirectional cascades of proteolytic enzymes of the blood clotting type and complement fixation. When triggered by an appropriate alarm signal the unidirectional cascade responds in an explosive manner to produce an avalanche of product needed to meet a specific biological emergency, but having fulfilled its function, the cascades are terminated by a self-destructive process that is initiated by autoregulatory signals. In effect, unidirectional cascades are contingency systems that function as biological ON-OFF switches in response to occasional emergency needs. The chapter also discusses the concepts that emerge, regarding cyclic cascades, as revealed by the simplified model. The capacity of biological systems to take advantage of the elegant regulatory mechanisms depends on the kinetic constraints, such as the concentrations and turnover numbers of the cascade enzymes and the dissociation constants of the effector–enzyme and enzyme–enzyme complexes. The kinetic analysis of the multicyclic cascade shows that, with reasonable parameters as stated above, a 50% modification of the second and third interconvertible enzymes in a three-cycle cascade can be accomplished in the millisecond time range. Further kinetic analysis shows that interconvertible enzyme cascade can in fact function as a rate amplifier to generate an almost explosive increase in enzymic activity in response to stimuli.

Subunit interaction of adenylylated glutamine synthetase.

The glutamine synthetase (GS) activity in Escherichia coli is regulated by a bicyclic interconvertible enzyme cascade which involves the cyclic adenylylation (inactivation) and deadenylylation (activation) of GS on the one hand, and the modulation of these processes by the uridylylation and deuridylylation of Shapiro's regulatory protein on the other. The specific activity of GS in a given metabolic state is determined by the fraction of its subunits that are adenylylated, and this fraction is determined by the concentration of over 40 metabolites. Through allosteric and substrate interactions with one or more of the cascade enzymes, these metabolites alter the rates of the covalent modification and demodification reactions. By means of immunoprecipitation studies with anti-AMP specific antibodies, it has been established that the partially adenylylated glutamine synthetase, which is present in a given steady state, is a mixture of hybrid molecules containing different numbers and possibly distributions of adenylylated subunits. Partial separation of these hybrid mixtures has been achieved by affinity chromatography on Affi-Blue Sepharose columns. From immunochemical studies it is evident that anti-AMP antibodies can react with adenylylated subunits of all molecular species of GS, but that the capacities of these primary antigen-antibody reactions to yield precipitable aggregates is very dependent of the number of adenylylated subunits per molecule, and much less so upon the total concentration of adenylylated subunits present. the studies suggest that precipitability is a function either of the distribution of adenylylated subunits within hybrid species, or of the kinetics of intra- vs intermolecular bivalent interactions.

Allosteric regulation of monocyclic interconvertible enzyme cascade systems: use of Escherichia coli glutamine synthetase as an experimental model.

The interconversion of Escherichia coli glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] between its adenylylated and unadenylylated forms has been used to verify the prediction derived from a theoretical analysis of the steady-state functions of a model for a monocyclic interconvertible enzyme cascade system [Stadtman, E. R. & Chock, P. B. (1977) Proc. Natl. Acad. Sci. USA 74, 2761-2770]. Because glutamine and alpha-ketoglutarate are multifunctional effectors and because three active enzyme complexes are involved in both adenylylation and deadenylylation of glutamine synthetase, at least 28 constants are required to describe the glutamine synthetase monocyclic cascade. Of these, 22 constants were determined experimentally and 6 were estimated via computer curve fitting. Despite the complexity, when both adenylylation and deadenylylation reactions are functioning, the number of adenylyl groups bound per mole of enzyme, n, assumes a steady-state level as is predicted by the model. This n value is determined by the mole fraction of P(IIA)-given by ([P(IIA)]/([P(IIA)] + [P(IID)])-and the ratio of glutamine to alpha-ketoglutarate (P(IID) and P(IID) are the unmodified and the uridylylated forms of the P(II) regulatory protein). In the presence of 0.5 mM glutamine and 2 mM alpha-ketoglutarate, the value of n increases as a nearly hyperbolic function in response to increasing mole fractions of P(IIA). When the constant level of alpha-ketoglutarate is gradually increased to 40 muM, the hyperbolic function converts slowly to a parabolic function. When the P(IIA) mole fraction was maintained at 0.6 and alpha-ketoglutarate levels were varied from 1 mM to 4 muM, an 800-fold increase in signal amplification was observed with respect to glutamine activation. In addition, because glutamine activates the adenylylation and inhibits the deadenylylation reaction, a sensitivity index of 2.1 (corresponding to a Hill number of 1.5) was obtained for the variation of n values in response to increasing glutamine concentration.

Interconvertible Enzyme Cascades in Metabolic Regulation

Publisher Summary This chapter focuses on interconvertible enzyme cascades in metabolic regulation. The cascades involved in the cyclic covalent modification of interconvertible enzymes are fundamentally different from the irreversible, unidirectional cascades of proteolytic enzymes such as are involved in blood coagulation and complement fixation. When triggered by appropriate stimuli, the latter cascades respond in an explosive manner to produce an avalanche of product needed to meet an occasional biological emergency. However, having fulfilled this function, the cascades are terminated abruptly in response to an entirely different set of autoregulatory signals that lead to self-destruction of the catalytic process. In effect, these unidirectional cascades are contingency systems that serve as biological switches that can be turned on or off intermittently to meet emergency situations. In contrast, the covalent modification of an interconvertible enzyme is a dynamic process in which the coupling of two opposing cascades leads to continual cyclic activation and inactivation of the enzyme. Such cascades are more properly designed for controlled amplification of primary stimuli, as is needed in the regulation of key enzymes in metabolism.

Superiority of interconvertible enzyme cascades in metabolite regulation: analysis of multicyclic systems.

Escherichia coli glutamine synthetase and glycogen phosphorylase are prototypes for models of "closed" and "opened" bicyclic cascade systems. Steady-state functions relating the fractional activation of interconvertible enzymes to the concentrations of allosteric effectors and to the catalytic constants of the several converter enzymes in such cascades were determined. The study shows that when the active form of an interconvertible enzyme in one cycle catalyzes the covalent modification of the interconvertible enzyme in a second cycle, the two cycles become coupled, such that the fractional activity of the second interconvertible enzyme is a multiplicative function of all parameters in both cycles, i.e., of 14 and 18 parameters for the closed and the opened bicyclic cascade, respectively. Therefore, from the standpoint of cellular regulation, bicyclic cascades are superior to the monocyclic cascades analyzed previously [E. R. Stadtman & P. B. Chock (1977) Proc. Natl. Acad. Sci. USA 74, 2761-2765], because: (i) they can respond to a greater number of allosteric effectors; (ii) they can achieve much greater amplification of responses to primary stimuli (e.g., with only 2-fold changes in each parameter the amplification factors of one-cycle and two-cycle cascades are 320 and 102,400, respectively); (iii) they can generate a sigmoidal response (Hill numbers of >2) of interconvertible enzyme activity to increasing concentrations of an allosteric effector. This is because there are more steps in a bicyclic cascade at which a given effector can interact. A similar analysis of multicyclic cascade systems shows that the capacity for amplification increases exponentially as the number of cycles in the cascade increases. In addition, regulation by cyclic cascades can achieve enormous variability of the fractional activity of the interconvertible enzyme by shifting the steady-state distribution between active and inactive forms. One equivalent of ATP is consumed in each interconversion cycle to provide the energy needed to maintain the steady-state activity of the modified enzyme at a metabolically required level. Therefore, the decomposition of ATP associated with the cyclic cascade is not a wasteful process.

Superiority of interconvertible enzyme cascades in metabolic regulation: analysis of monocyclic systems.

A theoretical analysis of monocyclic cascade models shows that the steady-state fraction of covalently modified interconvertible enzyme is a function of 10 different cascade parameters. Because each parameter can be varied independently, or several can be varied simultaneously, by single or multiple allosteric interactions of ligands with one or more of the cascade enzymes, interconvertible enzymes are exquisitely designed for the rigorous control of key metabolic steps. Compared with other reglatory enzymes, they can respond to a greater number of allosteric stimuli, they exhibit greater flexibility in overall control patterns, and they can generate a greatly amplified response to primary allosteric interactions of effectors with the converter enzymes. Contrary to earlier views, the decomposition of ATP associated with cyclic coupling of the covalent modification and demodification reactions is not a futile process. ATP decomposition supplies the energy needed to maintain concentrations of modified enzyme at steady-state levels that are in excess of those obtainable at true thermodynamic equilibrium.