Allosteric effectors enzyme sensitivity

Phosphorylation also can modify an enzyme s sensi-tivity to allosteric effectors. Phosphorylation of glycogen phosphorylase reduces its sensitivity to the allosteric activator adenosine monophosphate (AMP). Thus, a covalent modification triggered by an extracellular signal can override the influence of intracellular allosteric regulators. In other cases, variations in the concentrations of intracellular effectors can modify the response to the covalent modification, depending on the metabolic state of affairs in the cell. 

Connectivity theorems allow to relate the control coefficients (systemic properties) to the elasticity coefficients (properties of the network s enzymes individually as if in isolation) (Westerhoff and Van Dam 1987 Heinrich and Schuster 1996 Fell 1997). The connectivity theorems have given us a strong insight into the functioning of metabolic pathways. For example, it follows directly from these theorems that enzymes that are very sensitive to the concentrations of metabolites, such as substrates, products and allosteric effectors, tend to have little control over the flux. This is illustrated by overproduction of phosphofructokinase in bakers yeast, an enzyme often referred to textbooks as rate-limiting. Yet, overproduction of phosphofructokinase does not lead to a significant flux increase, since the cell compensates by lowering the level of its allosteric effector fructose 2,6-bisphosphate (Schaaff et al. 1989 Davies and Brindle 1992). 

The extreme sensitivity of the radical to oxygen explained why we lost it during purification and why the purified enzyme was inactive. However, with stricter adherence to anaerobic conditions, it was possible to prepare an enzyme that maintained a sizeable part of its glycyl radical. This was useful to identify the compounds of the system involved in the formation of the radical (activation) and those involved in the reduction of CTP. In fact, it appeared that CTP reduction by the radical-containing enzyme only required ATP, an allosteric effector, K+, Mg + and formate. As briefly described below, formate is the hydrogen donor during conversion of the ribose to deoxyribose [38]. 

RNR activity is readily detectable in soluble cell-free extracts of S. shibatae, S. solfataricus, and P. furiosus, incubated with cytidine diphosphate (CDP), dithio-threitol (DTT), and adenosylcobalamin (AdoCbl), at high temperatures (70°-80°). Sodium acetate stimulates the activity and is included in the assay. Acetate probably acts as an allosteric effector. The activity is not air-sensitive and purification can be achieved aerobically at room temperature, reflecting the extreme stability of the enzyme. The activity can be purified 3000-fold in three steps (ammonium sulfate precipitation, phenyl-Sepharose and dATP-Sepharose chromatography) (Table I). The key purification step is affinity chromatography on dATP-Sepharose (400-fold purification), a method used successfully for the purification of several other RNRs.3i.32 Binding to this ligand is an indication that the thermophilic RNR is... 

Correlations between critical temperatures for membrane lipid phases determined with EPR techniques and discontinuities in Arrhenius plots have also been shown for the ATPase from sarcoplasmic reticulum (Eletr and Inesi, 1972), for UDP-glucuronyltransferase, and for G-6-Pase (Eletr et al., 1973) from liver microsomes. In the case of the microsomal membranes, perturbation of the membrane lipids by treatment with detergents or phospholipase A leads to linear Arrhenius plots for both enzyme activities and Tq between 5° and 30 C. For UDP-glucuronyltransferase, the phase change in the lipids also results in a loss of substrate specificity and a loss of sensitivity to an allosteric effector (Vessey and Zakim, 1974). 

HMG-CoA reductase is also regulated by a reversible phosphorylation/ dephosphorylation cycle (Figure 7.17). The phosphorylated reductase is inactive and the amounts of the phosphorylated enzyme can be shown to be increased when its activity is decreased by mevalonate or glucagon. The protein kinase which inactivates HMG-CoA reductase is itself subject to phosphorylation. Interestingly, both ATP and ADP are needed. Apparently ADP binds to a different site on the reductase kinase and acts as an allosteric effector. The phosphatases which activate HMG-CoA reductase are highly sensitive to NaF. Both the kinase and phosphatases are present in both microsomal and cytoplasmic fractions. 

Phosphofructokinase activity is sensitive to both positive and negative allosterism. For instance, when ATP is present in abundance, a signal that the body has sufficient energy, it binds to an effector binding site on phosphofructokinase. This inhibits the activity of the enzyme and, thus, slows the entire pathway. An abundance of AMP (adenosine monophosphate), which is a precursor of ATP, is evidence that the body needs to make ATP to have a sufficient energy supply. When AMP binds to an effector binding site on phosphofructokinase, enzyme activity is increased, speeding up the reaction and the entire pathway. 

In contrast with Michaelian enzymes, which have hyperbolic kinetics, allosteric enzymes, thanks to their sigmoidal kinetics, possess an enhanced sensitivity towards variations in the concentration of an effector or of the substrate. This is the reason why many enzymes that play an important role in the control of metabolism are of the allosteric type. 

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