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7.7Regulation of Enzymatic Activity

7.7.1

Regulation of Enzymatic Activity

Enzymatic activity is controlled at the enzyme content level through the synthesis and degradation of enzymes as well as at the level of individual enzymatic molecules by low molecular substances (effectors) such as metabolites, ATP and ADP. When effectors are attached to sites other than the active centers of enzymes, these sites are known as allosteric sites, and the enzymes regulated by them are called allosteric enzymes (allo means “other” and steric means “space”). Phosphofructokinase (PFK) - a major enzyme in glycolysis - is a typical allosteric enzyme (Fig. 7-4), and is inhibited by ATP and citric acid and activated by AMP, ADM and fructose 6-phosphate.
Generally, the activity of allosteric enzymes follows a sigmoid curve against substrate concentration and effector concentration, and is greatly influenced by small changes in effector concentration (Fig. 7-5A). Although enzymes have a steric structure, such activity regulation is difficult for monomeric enzymes. This mechanism of allowing activity regulation, like turning a switch on and off, is explained by cooperative structural changes in polymers. Generally, allosteric enzymes are polymers consisting of many subunits, and have steric symmetry. It is not possible for only one subunit to change its structure, but all subunits can simultaneously take slightly different structures. It has therefore been postulated that two conformations - T and R - are at equilibrium (Fig. 7-5B). Here, it is assumed that T-subunits are less active than R-subunits, T-subunits have a higher affinity for effectors, and R-subunits are bound to the substrate more firmly. Although T-subunits, with their inability to change structure readily, are slow to bind to substrate with increased substrate concentration, once a certain concentration is exceeded, their activity increases in the shape of a sigmoid curve. Similarly, R-subunits are slow to bind to effectors while the effector concentration is low, but once a specific threshold is exceeded, they are simultaneously bound to effectors and are transformed into T-subunits, thus significantly lowering enzymatic activity.
This is only an outline, and a number of models have been proposed that demonstrate the concept in further detail. In addition, actual allosteric enzymes are rather complex, and consist of several types of subunit. As an example, pyruvic acid dehydrogenase consists of a large number of three subunit types: E1, E2 and E3 (60 subunits in the case of E. coli).

Fig. 7-5. Model of cooperative structural change in allosteric enzymes

A) The binding between an allosteric enzyme and a substrate follows a sigmoid curve, which moves from side to side depending on the effector concentration, thereby regulating enzymatic activity.
B) A cooperative structural change model for allosteric enzymes. Such enzymes are generally polymers, and the strength of activity is varied (as if turning a switch on and off) through cooperative structural changes in all subunits.

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Metabolomics

The quantification of all metabolites in a cell would enable the functioning of cellular metabolic pathways to be modeled; this in turn would allow clarification of whether the metabolic mechanism can be explained by known regulatory mechanisms only, or whether different regulatory mechanisms are at work. Based on this concept, the field of metabolomics aims to identify types of metabolites and determine their quantity as far as possible by using computers to automatically perform chromatography, electrophoresis and mass spectrometry. Currently, the number of materials that can be analyzed is still limited, and significant improvement in analytical techniques is necessary.

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7.7.2

Enzymatic Activity Regulation by Phosphorylation

Aside from allosteric regulation, enzymatic activity is also regulated through enzyme-molecule-level modification by covalent bonds. The phosphorylation involved in signal transduction (see Chapter 9) is a typical example of this. In the phosphorylation of proteins, a phosphoester bond is formed in the hydroxyl group of serine, threonine or tyrosine residue. Protein kinases with varying levels of specificity exist depending on which side chain of which protein is phosphorylated by the enzyme. Normally, phosphorylation takes place using ATP as a substrate, and during this process the phosphate group at the terminal is transferred to proteins. Phosphoprotein phosphatase - an enzyme that hydrolyzes phosphoester bonds in each protein - is also found. The activity balance of the two enzymes regulates the activity of the target enzymes.
Pyruvate dehydrogenase (PDH) is allosterically inhibited by acetyl-CoA and NADH, and its enzymatic activity is lowered by phosphorylation and recovered by dephosphorylation. Protein phosphorylation enzymes that catalyze this phosphorylation are activated by acetyl-CoA, NADH and ATP (broadly speaking, substances generated by the PDH reaction) and inhibited by pyruvic acid, CoA-SH and NAD+ (broadly speaking, substances accumulated when the PDH reaction is insufficient). Phosphatases that catalyze this dephosphorylation are activated by the stimuli of Ca2+. In the case of PDH, such phosphorylation-mediated regulation is also a type of feedback.
Since phosphoenzymes are themselves regulated by phosphorylation, multi-stage phosphorylation cascades are sometimes formed; the regulation of blood sugar levels via hormone-mediated control of glycolysis and gluconeogenesis is a typical example of this. The name “cascade” expresses a consecutive series of reactions occurring in the same way as running water pouring over a cliff.
Figure 7-6 shows phosphorylation cascades describing how blood sugar levels are raised by glycogenolysis, which is controlled by hormones and is well known for its occurrence in the liver and muscles of humans. Signals from hormones such as adrenaline and glucagon are transformed to cAMP (see Chapters 2 and 9) in cells before being transmitted to phosphorylation enzymes. During the following cascades, glycogen phosphorylase b is activated, breaking down glycogen and thus supplying large amounts of glucose to blood and muscle tissues for use. In the liver, the phosphofructokinase and pyruvate kinase (Fig. 7-4) involved in glycolysis are also inhibited by cAMP, prompting the release of undegraded glucose to the blood. Insulin, on the other hand, lowers blood sugar levels and promotes glycogen synthesis.

Fig. 7-6. Cascades of carbohydrate mobilization regulation by a hormone (adrenalin)

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7.7.3

A Paradigm of Metabolic Regulation: Feedback Regulation and Cascades

One of the basic regulation mechanisms of metabolism is feedback regulation, in which a metabolic pathway is stopped when the amount of final product becomes excessive. With such regulation, not all enzymes involved in the pathway need to be inhibited, and it is common for the most important enzyme in the pathway to be inhibited instead. In glycolysis, phosphofructokinase is the main control point of feedback regulation (Fig. 7-4). As a result, allosteric inhibition by ATP and citric acid (generated by glycolysis and in the subsequent citric acid cycle and by oxidative phosphorylation) takes place against the enzyme. Additionally, in the case of regulation by phosphorylation of pyruvic acid dehydrogenase, the activity of phosphoenzymes is allosterically inhibited by ATP and citric acid (the final products), establishing feedback regulation as a result.
The term feedback (or negative feedback) originally comes from system engineering, and refers to a mechanism by which the output of a system is kept constant by continually converting the output to a negative signal and returning it to the input. If the output is returned to the input without this conversion, the system will diffuse. Although positive feedback is rare in biological systems, nerve impulse generation is an example of this (see Column Fig. 5-3 in Chapter 5). On the other hand, the cascades outlined previously represent a system that amplifies input through multiple stages, and function to create a profound effect by using small inputs in the same way as switches. Linking an action system with nonlinear responses (such as allosteric regulation) to this enables all-or-none signal regulation. In many biological phenomena, inputs are continuous variables such as environmental conditions, whereas outputs are sometimes all-or-none responses such as cell differentiation. By further devising a combination of basic regulation models, such nonlinear responses can be analyzed.

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Why are Metabolic Pathways Circular?

As we have already seen, metabolic pathways (including the citric acid cycle, the Calvin cycle and the urea cycle) are often circular. Why is this? In a round metabolic cycle, when the ∆G value of reaction A is near zero, thus bringing it close to equilibrium, the concentration of the reactant in reaction A increases as other reactions proceed. This moves the equilibrium of reaction A to the reactant side, thus pushing it to proceed. When a cycle turns to a steady state, the rates of all the constituent reactions are made equal by keeping the concentration of each intermediate at an appropriate level. This is the secret of the circular nature of many metabolic pathways, in which reactions that are slow to proceed are supported by other reactions that are fast, thus proceeding the reaction system along smoothly.
This means that the progress of reaction A is regulated by other reactions. As an example, the citric acid cycle halts if the electron transport chain stops, since succinate dehydrogenase is coupled with the electron transport chain. While allosteric regulation is the regulation of a reaction by effectors at the single-enzyme level, metabolic cycles as a whole have a regulation system similar to that of allosteric regulation. Furthermore, most reactions, including the transformation between ATP and ADP, have normal and reverse reactions, thereby forming cycles. This means that reactions within cells are engaged and influenced by each other like gears. In the same way as a mill that grinds flour by passing water flow over a wheel, various metabolic cycles are turned by the degradation flow of energy from nutrients, thereby allowing the activities of cells and organisms to take place.

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