Chapter 12—
Regulation of Enzyme Levels and Activity
12.1—
Introduction
The first theory of metabolic control was introduced into plant physiology in 1905 by F. F. Blackman and has become known as the 'Law of Limiting Factors' which states that 'when a process is conditioned as to its rapidity by a number of separate factors the rate of the process is limited by the pace of the slowest reaction'.
It is not possible to derive a rate equation from this statement and discussions of the 'Law' tend to be by analogy, e.g. the strength of a chain is the strength of its weakest link. Experimentally, the statement has led to the fruitless search for metabolic master reactions. Mathematically, we can derive rate equations for multistep reactions and establish that every step contributes to the overall rate. Theoretically Blackman's statement is a denial of the steady state; if a metabolic sequence is in a steady state then the concentration of each intermediate is constant and the individual reactions proceed at the same rate—hence no reaction can be described as the slowest.
12.1.1—
Pacemakers
If in a metabolic sequence all reactions are proceeding at the same pace then a single reaction may be almost entirely responsible for determining that pace; such a reaction has been called a 'bottleneck' or pacemaker and much work in metabolic control is directed towards the identification of such control points. Krebs and Kornberg (1957) in a consideration of pacemakers stated 'there is a principle which may guide the search for pacemakers. As pacemakers are reactions of variable rate, the level of substrate concentration of the pacemaker must vary inversely with the rate: it must increase when the reaction rate decreases'. It should be possible to take published data for a metabolic pathway, apply the Krebs-Kornberg principle and so determine which reaction is the pacemaker. It turns out that in some cases the Krebs-Kornberg principle cannot be applied. For example in some tissues when the rate of glycolysis is increased not a single compound shows the expected reduction in concentration.
12.1.2—
Occam's Razor
William of Occam, the mediaeval controversialist, formulated a procedural rule, that when a number of possible solutions can be proposed for a problem,
one should accept the simplest solution until it is shown to be untenable. The Krebs-Kornberg principle is a simple solution to a complex problem and in certain cases it yields a valid solution. However, when it is not applicable we must examine more complex solutions.
12.1.3—
Systems Properties
The rate equation for a metabolic pathway includes parameters for all the enzymes and variables for all the metabolites (Waley, 1964). The flux through the pathway is a systemic property in which all the parameters interact as a system. If a single parameter is altered, say the activity of a single enzyme, then the whole system responds and adjusts to that change. The extent to which an enzyme can be considered a control step is the extent to which a fractional change in its activity produces a fractional change in the flux through the system. The ratio
has been termed the sensitivity coefficient for that step (Kacser & Burns, 1973). If a 1 % change in activity of an enzyme produces a 1% change in flux through the system, then the sensitivity coefficient is 1 and the enzyme must be fully controlling the flux and is a pacemaker. However, the sum of all the sensitivity coefficients for a metabolic sequence is equal to unity and only in special circumstances can a single pacemaker be identified. That the sensitivity co-efficient of a particular enzyme is a system property, only in part determined by its own parameters, can be intuitively understood by an example. In a given situation an enzyme may have a very small sensitivity coefficient and contribute little to the overall control of the system. If however the activity of this enzyme is drastically reduced its sensitivity coefficient may be increased and it may contribute significantly to the overall flux. Since the sum of sensitivity coefficients must always equal unit the sensitivity coefficients of all the other enzymes must have changed despite the fact that their parameters are unchanged.
Much work on metabolic control is concerned with the identification of control points. Since each reaction contributes to the overall control a sense of judgement is necessary to identify major control points and it must be remembered that control may pass from one reaction to another as the flux through the system changes.
12.2—
The Identification of Control Points
12.2.1—
Equilibrium Considerations
Bücher and Russmann (1964) have proposed a model for metabolic control based on a controlled waterway (Fig. 12.1).
Figure 12.1
Schematic representation of a regulated waterway. The solid line represents the
situation when the flood gates are raised and there is little flow through the system.
The dashed line represents the increased flow when the flood gates are lowered.
The flow of water through the system is controlled by the flood gates. With a flood gate up, there is a negligible gradient across the reservoir and a slight flow of water over the dam. In metabolic terms the reaction 
represents the reservoir and can be considered as a fast reaction close to equilibrium 
. The reaction B® C represents the waterfall and is a slow reaction far from equilibrium (D G is large).
When the flood gate is lowered a slight gradient is produced across the reservoir and there is a rapid flow of water over the dam, which could be utilized to perform work. In metabolic terms, as the flux through the system increases, the reaction moves away from equilibrium and the controlled reaction B ® C increases in rate and simultaneously moves towards equilibrium. The model thus identifies control reactions as reactions far from equilibrium and which move towards equilibrium as the flux increases. Since D G for such reactions is large, control points will tend to be associated with reactions involved in the production of useful work by a coupled reaction. However, the useful work function is not an essential feature of the model and in the absence of a coupled reaction to utilize the large D G, the loss of energy can be considered as the price paid for control.
12.2.2—
The Crossover Theorem
The crossover theorem states that: the variations of the concentrations of the metabolites before and after an enzyme which is a control point have different signs. It will be noted that this is only a formal statement of the situation at the waterfall in the Bücher and Russmann analogy, i.e. when the flux increases the concentration of the substrate falls and the concentration of the product
increases. However, the theorem is widely used, possibly because it presents data in a graphical form. Metabolic intermediates are plotted on the x coordinate in the sequence in which they are formed in the metabolic pathway and the concentration of each substrate, expressed as a percentage of the initial value is plotted on the y coordinate. An example is shown in Fig. 12.2 for the situation usually referred to as the 'Pasteur Effect'. Pasteur observed that 'oxygen inhibits fermentation' so that when carrot discs are transferred from air to nitrogen glycolysis increases with associated changes in the levels of intermediates. These changes are plotted for a period some 6 minutes after transfer from air to nitrogen (Fig. 12.2).
Figure 12.2
Crossover plot of glycolytic intermediates following transfer of
aged carrot discs from air to nitrogen (Data of Faiz-ur-Rahman,
Trewavas & Davies, 1974). Intermediates were measured before
transfer and 6 minutes after transfer from air to nitrogen.
The positive crossover observed between fructose-6-phosphate and fructose diphosphate suggests that phosphofructokinase is a control point. There is some confusion concerning the interpretation of negative crossovers. Some authors argue that a negative crossover does not indicate a control point. However, it must be remembered that when the flux through a system is changed, control can pass from one point to another. Thus in the case illustrated in Fig. 12.2 the increased flux through the glycolytic system involves an increase in activity of phosphofructokinase but with the increased rate of glycolysis, phosphoglycerate is removed more rapidly than it is formed, suggesting that a bottleneck exists between dihydroxyacetone phosphate and phosphoglycerate when phosphofructokinase is highly active.
The crossover theorem should be used with caution; the absence of a crossover should not be taken to mean the absence of a control point and there are many conditions which can produce crossovers indicating spurious control points. Before applying the crossover theorem, the reader is advised to consult the critique offered by Heinrich and Rapoport (1974) and to remember that
control is a property of the system; the identification of a control point is to focus attention on one component knowing that to varying degrees all other enzymes in the sequence contribute to the overall control.
12.3—
Control Mechanisms
Having identified possible control points it becomes necessary to determine the mechanism of control. Over a relatively long time scale control may be achieved by changing the amount of an enzyme but over a short time scale control is likely to be achieved by changing the activity of the enzyme.
12.3.1—
Control by Product Inhibition
Every enzyme has a certain affinity for its products and if the enzyme has a high affinity for its product then product inhibition will be pronounced. It appears likely that this form of control is restricted to some synthetic routes and to minor metabolic sequences.
12.3.2—
Control by Negative Feedback
The discovery of negative feedback in metabolic systems appears to have been made by Dische in 1940 who noted that 3-phosphoglycerate inhibits the phosphorylation of glucose in red cells and he proposed that this inhibition might play a regulatory function in glucose metabolism. His paper did not receive the attention it deserved and the idea of feedback had to be rediscovered.
In 1955 Roberts and his coworkers published the result of their extensive studies of the metabolism of E. coli grown on a medium containing 14 C-glucose. They found that the addition of one of a number of amino acids (known to be products of specific synthetic pathways) to the growth medium resulted in the virtually complete inhibition of incorporation of 14 C into that amino acid.
In 1956 Umbarger reported that isoleucine inhibited threonine deaminase—an enzyme which initiates the set of reactions leading to isoleucine biosynthesis. In the same year Yates and Pardee observed that cytidine monophosphate inhibits aspartate transcarbamylase—the first reaction of the pathway leading to the biosynthesis of the pyrimidines. These results offered a biochemical explanation for the end-product inhibition observed by Roberts with intact bacteria.
In 1959 Sir Hans Krebs introduced the first symposium to be held on metabolic control. Ideas on the regulation of carbohydrate and amino acid metabolism were discussed and the general ideas of feedback formulated. Control by negative feedback is an engineering concept and familiar examples include governers of steam engines, thermostats and automatic volume control in amplifiers. The limitations of the analogy between electrical feedback amplifiers and metabolic reactions have been stressed by Britton-Chance. However,
as with other terminology developed for use with metabolic control systems (see discussion of allosteric enzymes) the tendency has been to dispense with rigorous definitions and the general definition of negative feedback is 'increased output decreases the input'.
12.3.3—
Patterns of Control
The most frequently observed pattern of control is when the end. product of a metabolic sequence inhibits the first reaction belonging to that sequence. For example serine can be formed by the reaction sequence shown in Fig. 12.3.
Figure 12.3
Reactions involved in the biosynthesis of serine.
Serine inhibits 3-phosphoglycerate dehydrogenase and so controls its own biosynthesis. This simple mechanism is effective, presumably because a relatively small amount of the flux through the glycolytic pathway is directed towards serine and thus variations in the rate of serine production will not seriously perturb the glycolytic flux.
12.3.4—
Control of Branched Pathways
Inhibition of the first step in a metabolic pathway by an end-product leads to special problems in the case of branched biosynthetic pathways. Consider the sequence
It the first common step (A® B) is inhibited by either or both end-products, then an excess of one could inhibit the step A® B and lead to a deficiency of the
other end-product. Nature has evolved several mechanisms which avoid, to varying degrees, these difficulties. Stadtman (1970) has classified these mechanisms; here we discuss a few examples which have been studied in plants.
12.3.4.1—
The Aspartate Family
The formation of some amino acids from aspartate is outlined in Fig. 12.4 together with feedback controls which have been demonstrated.
Figure 12.4
Reactions involved in the biosynthesis of the aspartate family of amino acids.
® Single step reaction; ---® multi-step reaction; --® feed back loop.
Aspartokinase which catalyses the first reaction of the pathway is inhibited by lysine and threonine in a concerted or synergistic manner. The term concerted feedback inhibition has been used by Stadtman to describe cases of inhibition by two end products when the single end products produce no inhibition. The term synergistic inhibition is used when the total inhibition is much greater than the sum of their independent effects. The aspartokinase from plants appears to be inhibited by low concentrations of lysine but not by low concentrations of threonine—when both are present a much greater inhibition is observed. Although this effect would seem best to fit Stadtman's definition of synergistic inhibition it has been called concerted inhibition. Whatever the terminology, this mechanism does not involve methionine and is thus only a partial solution to the control of the branched pathway. The non-involvement of methionine means that in conditions where threonine and lysine
accumulate they will tend to inhibit the synthesis of methionine—even in situations where methionine may be in short supply. This imperfection is compounded by the feedback inhibition of homoserine dehydrogenase by threonine which further reduces the flow of intermediates available for the biosynthesis of methionine.
These imperfections in the control mechanism can be invoked to explain the effect of amino acids on the growth of Lemna minor (Table 12.1).
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The relatively small inhibition observed with lysine can be interpreted in terms of inhibition of aspartokinase (but see section on metabolic interlock ). The inhibition produced by threonine is consistent with the inhibition of homoserine dehydrogenase which would prevent the flow of carbon to methionine. The inhibition produced by homoserine is difficult to explain since as far as known it does not inhibit any of the enzymes involved. Furthermore in some plants e.g. peas, homoserine is produced in large quantities and transported in the phoem. The inhibition produced by isoleucine is consistent with a control pattern known as sequential inhibition. Isoleucine has been shown to inhibit threonine deaminase which would be expected to lead to an accumulation of threonine which in turn inhibits homoserine dehydrogenase and so reduces the flow of carbon to methionine. The inhibition produced by methionine is difficult to explain in terms of the reactions shown in Fig. 12.4. The pronounced inhibition observed with lysine and threonine is consistent with their synergistic effects on aspartokinase producing a reduction in the flow of carbon to methionine, which is reinforced by the inhibition of homoserine dehydrogenase
by threonine. Aspartic acid and isoleucine are unable to reduce the inhibition but methionine and homoserine are able to do so, presumably by supplying methionine directly or indirectly.
12.3.4.2—
Aromatic Biosynthesis
The control of aromatic amino acid biosynthesis has been extensively studied in fungi and bacteria and it is clear that nature has evolved many solutions to the problem of control in branched pathways. Some of the solutions which have been demonstrated in higher plants are shown in Fig. 12.5.
12.3.4.3—
Enzyme Multiplicity
In this control mechanism the first common step is catalysed by two or more enzymes which are under feedback control by compounds formed after the branching point. Four examples are given in Fig. 12.5, the control of chorismate mutase being particularly complicated. Three isozymes have been demonstrated in plants (Woodin & Nishioka, 1973); CM1 and CM3 are inhibited by phenylalanine and activated by tryptophane. CM1 and CM2 are inhibited by caffeic acid and chlorogenic acid whilst CM3 is inhibited by ferulic acid.
12.3.4.4—
Enzyme Aggregation
A number of fungi possess an aggregate of the five enzymes necessary for the conversion of 3-deoxy-D -heptulosonate-7-phosphate to enolpyruvyl-shikimate-5-phosphate. In Neurospora the five enzymes form a complex (M.W. 230,000) coded by the arom gene cluster. This large complex has not been demonstrated in higher plants, but a bifunctional enzyme consisting of dehydroquinase and shikimate dehydrogenase has been isolated (Boudet, 1971). Neurospora contains two dehydroquinases, the one in the aggregate is involved in an anabolic sequence, the other maps outside the arom cluster and is thought to function as a component of a catabolic sequence. The physiological significance of the multienzyme cluster could be to separate the degradative and synthetic routes. This could be achieved if the dehydroquinic acid which is formed in the aggregate is not released as a free product. This idea has become known as metabolic channelling and in a number of cases it has been shown that metabolites involved in channelling do not leave the enzyme surface. For example fungi possess a bifunctional enzyme consisting of carbamyl phosphate synthetase and aspartate transcarbamylase.
Figure 12.5
Reactions involved in the biosynthesis of aromatic compounds.
® Single step reactions; --® multi-step reactions; --® feed back loop.
When the bifunctional enzyme is synthesizing ureidosuccinate from 14 CO2 , the addition of carbamyl phosphate does not dilute the incorporation of 14 C into ureidosuccinate, showing that carbamyl phosphate formed by the double enzyme does not equilibrate with free carbamyl phosphate.
Higher plants contain two dehydroquinases one forming a bifunctional association with shikimate dehydrogenase, the other being activated by shikimic
acid. Such a system involving a specific regulation of isoenzymes and molecular compartmentalization may function to control the partitioning of dehydroquinic acid between the pathways to phenolcarboxylic acids and aromatic amino acids.
12.3.5—
Metabolic Interlock
The reactions and feedback loops shown in Fig. 12.5 give some idea of the complexity of control within a metabolic pathway. When the feedback loops of other metabolic pathways are taken into consideration it seems highly likely that the control must be interlocked. As an example, consider the reactions involved in the biosynthesis of methionine. In Fig. 12.4, this is shown as the addition of a C1 group to homocysteine. The synthesis of this C1 group is shown in Fig. 12.6.
Figure 12.6
Reactions involved in the biosynthesis of methionine.
® Single step reaction; ---® multi-step reaction; . . .® feed back loop.
The active C1 group is generated in the serine hydroxymethyl transferase reaction as methylene tetrahydrofolate. After reduction to methyl THFA the methyl group is transferred to homocysteine to form methionine. Homocysteine is derived from the aspartate family (cf. Fig. 12.4) and so is lysine. The metabolism of the aspartate family and the serine glycine family are thus interlocked via lysine, which inhibits both aspartokinase and serine hydroxymethyl transferase.
12.3.6—
Enzymes as Control Elements
Until the discovery of feedback inhibition in 1956 enzymes were considered as biological catalysts and most enzymologists were concerned with the properties of the active site. When it became apparent that enzyme activity was under the control of specific metabolites, which were structurally unlike the active site, it became necessary to postulate a control site to which metabolites could bind and modulate the activity of the enzyme. Recognizing the lack of structural similarity between the substrate and the effector the term allosteric site was introduced (Monod, Changeux & Jacob, 1963). Subsequently Monod, Wyman and Changeux (1965) noted that many regulatory enzymes with specific allosteric sites exhibit cooperative-type kinetics and they developed a concerted transition hypothesis to explain the observed kinetics. Many workers assume that any enzyme showing sigmoidal kinetics is an allosteric enzyme and some workers imply that the mechanism of activation of all allosteric enzymes follows the mechanism postulated by Monod et al., (1965). The purist may object but the original precise definition has been replaced by a broader but less precise usage.
12.3.6.1—
Allosteric Enzymes
Before 1956, the kinetics of numerous enzymes were shown to follow the rate law of Michaelis and Menten giving a hyperbolic plot of v against S . An examination of enzymes which were known to be subject to feedback regulation showed that in some cases the relationship between v and S was sigmoid. In other cases the addition of a feedback inhibitor led to a change from a hyperbolic to a sigmoid relationship.
Figure 12.7
Illustration of the Monod model of an allosteric enzyme.
Monod et al., (1965) proposed a simple model to account for the observed kinetics in which they took account of the observation that allosteric enzymes are composed of subunits. They assumed that the protein subunits were arranged in such a way that they occupy equivalent positions so that the enzyme must have at least one axis of symmetry. They further assumed two interconvertible states—R and T—each maintaining the symmetry principle. A simple illustration of the model is given in Fig. 12.7 based on the symbols used by Koshland (1970). The reader is referred to the original article by Monod et al., (1965) for the derivation of the rate law. However a consideration of the illustration leads to a basic understanding of the underlying causes of sigmoid kinetics. Two types of allosteric behaviour may be distinguished—one the variable K system in which the two states of the enzyme have different affinities for the substrate, the other, the variable V system in which the R state is catalytically more active (Fig. 12.8).
Figure 12.8
Relationship between v and s.
(A) a variable K system; (B) a variable V system.
Variable K Systems
In the variable K system, the R form is assumed to have a higher affinity for S than the T form of the enzyme. If in the absence of ligands, the equilibrium favours the T form then increasing concentrations of S will shift the equilibrium towards the R state giving sigmoid v against S plots. Another way of looking at this situation is to imagine that the interconversion of the R and T forms can be blocked and the enzyme isolated as the R or T form. The T form would give a normal Michaelis-Menten curve with a high Km(T ) and the R form would also give a Michaelis-Menten curve but with a low Km(R ) . Allowing R and T to be interconvertible allows Km to vary.
If an activator binds preferentially to the R state it will tend to put the enzyme entirely in the R state and so remove the sigmoidicity of the v against S plot. Similarly if an inhibitor binds preferentially to the T form it will increase the sigmoidicity.
Variable V Systems
In the variable V system the R form is assumed to be catalytically more active than the T form though both forms have the same affinity for the substrate so that the v/S plot is hyperbolic in the presence or absence of activators and inhibitors. An activator is assumed to have a greater affinity for the R form than for the T form whilst the reverse holds for an inhibitor.
12.3.6.2—
Alternative Models for Allosteric Enzymes
This superficial consideration of the Monod model concentrates on only one aspect of the model–the two configurational states. This limited aspect gives a qualitative explanation of' sigmoid kinetics described by the equation:
where N = 2.
To explain higher order reactions the Monod model includes some highly restrictive assumptions which produce mathematical simplicity but are nevertheless based on the fundamental properties of protein structure.
An alternative model proposed by Koshland (1970) assumes an induced fit so that the protein subunit undergoes a conformational change when it binds a ligand. A simple sequential model may be illustrated:
This simple model is not a necessary part of the induced fit model (which in fact suggests a diversity of forms of association between enzyme and ligand—25 for a tetrameric protein). Nevertheless it simplifies the mathematical treatment and in many cases provides a close fit to experimental values.
12.3.6.3—
Kinetic Constants for Allosteric Enzymes
When an electronic engineer constructs a control circuit he needs to know the characteristics of the components—the amplification factor, grid bias, etc. Similarly if we wish to understand the control of a system we need to know the characteristics of its components, that is, the kinetic constants of the enzymes. Clearly a variable K system cannot be defined by a Km value nevertheless the same operation value can be used, that is the concentration of substrate producing half maximum velocity which is in this case designated S0.5 . More generally the term (X0.5 ) is used to designate the ligand concentration at half saturation.
The constant N is operationally the order of the reaction, its physical meaning may relate to the number of substrate binding sites on the enzyme or to the degree of cooperativity of binding. Whatever the meaning of N it can be estimated by means of a Hill plot (Fig. 12.9). Another kinetic term, Rs is the ratio between the 90% and 10% saturation values. Rs will be 81 for all curves following Michaelis-Menten kinetics and less than 81 for cases of positive cooperativity–a few cases of negative cooperativity are known and these give Rs values greater than 81.
Figure 12.9
Kinetic data for an enzyme with variable 'K' properties.
The curves are drawn assuming 
. (A)N = 2; (B) N = 4.
Valve characteristics define the properties of the valve but do not determine the function of the valve–the function of the valve is determined by the circuit in which it operates, in one circuit it may function as a cathode follower, in another as an amplifier. Similarly, the kinetic constants of a control enzyme do not determine its role in metabolic organization. The properties of an enzyme in a metabolic pathway are modified by its association with other enzymes. The study of control in metabolic pathways relates enzymology to physiology and illustrates the somewhat unfashionable view that the whole is more than the sum of the parts.
12.4—
Control Mechanisms Involving Changes in the Amount of Enzyme
The brilliant work of Jacob and Monod (1961) on the regulatory mechanisms governing b -galactosidase activity in E. coli led to many attempts to demonstrate similar control systems in plants. A long series of negative experiments has made it unlikely that operons exist in higher plants. There is, however, evidence for the interdependent regulation of some groups of enzymes. For example in parsley and soybean cell cultures, the three enzymes catalysing the synthesis of p -coumaroyl CoA from phenylalanine show strictly concomitant changes in activity. Large changes in activity are initiated by light but there is no satisfactory explanation for the strict coordination in activity. Mohr (1972) has suggested that genes can be divided into four classes—active genes which function in the same way in light and dark, inactive genes which function only at special times in the life history of the plant (e.g. flowering genes), potentially active genes whose activation is under the control of the phytochrome system and repressible genes which can be repressed by the phytochrome system. Positive photoresponses are identified with gene activation and negative photoresponses with gene repression. This model is so basic that it must contain some truth, but it offers no explanation of the mechanism of regulation other than to identify regulation as occurring at the gene level. Much of the evidence for Mohr's model is concerned with the increase in phenylalanine ammonia lyase which occurs when mustard cotyledons are exposed to far-red light. Recently however, it has been suggested (Attridge et al., 1974) that this increase in activity is due to an activation of pre-existing enzyme rather than synthesis.
Following the success in understanding the regulatory system in prokaryotes it was clearly sensible to see if the system was applicable to plants. It now appears that the regulatory systems are very different, but the technical vocabulary used in the study of prokaryotes is still used in studies with plants. It should be noted that terms such as repression and derepression have precise meanings in relation to molecular models of regulation in prokaryotes, but when applied to plants, these terms should not be taken to imply a mechanism similar to that found in bacteria.
Many cases are known where a metabolite reduces the level of an enzyme involved in its metabolism (Table 12.2). Most of these cases are reported as
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examples of repression but the mechanism involved varies from case to case—for example arginine probably exerts its effect by stimulating proteolysis, the effect of ammonia may be due to proteolysis, though the formation of a specific protein inactivator has been proposed, and the effect of glucose on invertase has been reported as due to the destruction of m-RNA required for invertase synthesis.
The regulation of enzyme levels may involve one or more of the following:
(a) Degradation
(b) Inactivation
(c) Activation
(d) Synthesis
When examining a particular system it is necessary to establish which control system is involved and to measure its rate—this is frequently surprisingly difficult.
12.4.1—
Protein Turnover and Degradation
The view that plant proteins may undergo a cycle of synthesis and degradation was first proposed by Gregory and Sen in 1937. The terminology is somewhat confused; protein turnover implies synthesis and degradation but usually only degradation is measured.
Three methods are available for determining the overall rate of protein degradation.
(a) Incorporation of 14 C-amino acids.
The tissue is pulse-labelled with a 14 C-amino acid and the incorporation into protein measured. During the chase period the rate of protein degradation is estimated from the fall of radioactivity in the protein. The method has two serious disadvantages: (1) The recycling of protein amino acids—that is the reincorporation of a 14 C-amino acid released by degradation tends to underestimate the rate of degradation. This disadvantage can be minimized by using a 14 C-amino acid which is rapidly metabolized; (2) The existence of pools of amino acids means that the specific activity of the amino acid at the site of protein synthesis is unknown. Furthermore the incorporation of a labelled amino acid into a metabolic pool during the pulse period may be followed by a slow release of 14 C-amino acid during the chase. This disadvantage can be minimized by using an amino acid whose pool size is small, e.g. leucine.
(b) Measurement of the specific activity of aminoacyl t-RNA.
Trewavas (1969) has developed a method in which the specific activity of the amino acid entering protein during a labelling experiment is determined by isolating the appropriate aminoacyl t-RNA.
(c) Measurement of the incorporation of 3 H.
Humphrey and Davies (1975) established that when Lemna fronds are exposed to 3 H2 O, the tritium rapidly enters and equilibrates with H on the a -carbon atoms of amino acids. Protein synthesized during the exposure to 3 H2 O is thus
labelled in a stable position. When the plants are transferred to H2 O the amino acids rapidly lose their 3 H, the 3 H in protein is stable, but as soon as the protein is degraded to amino acids the 3 H exchanges with H2 O. Thus the rate of protein degradation can be determined simply by measuring the loss of 3 H from protein. The method depends upon the speed with which transaminases catalyse the exchange reaction between water and the hydrogen on the a -carbon atom of amino acids. If it can be established for a particular tissue that this reaction is fast relative to the rate of protein synthesis the method overcomes problems of pools and recycling. The method can in principle be applied to a specific enzyme, but this requires the isolation of the pure enzyme.
12.4.1.1—
Degradation of Specific Enzymes
If the degradation of proteins were a random process then a low rate of protein hydrolysis would almost completely destroy overall enzyme activity. This is because a 5% per day rate of degradation means that each enzyme molecule would be subject to an average degradation of 5% and for many enzymes this would mean a complete loss of catalytic activity. Consequently we must look for specificity in degradation.
A NADH-nitrate reductase-inactivating enzyme has been isolated from maize roots which appears to have a high degree of specificity (Wallace, 1974). The enzyme has been purified 460-fold, the purified preparation has proteolytic activity but no peptidase activity and the capacity to inactivate nitrate reductase is blocked by phenylmethylsulphonyl fluoride suggesting the involvement of a serine residue at the active site.
12.4.1.2—
The Measurement of Enzyme Half-Lives
If protein synthesis is blocked by an appropriate inhibitor and at various time intervals the level of enzyme is measured, we can determine the fall in activity of the enzyme. If we assume that the inhibitor completely blocks protein synthesis without interfering with the reactions involved in inactivating or degrading the enzyme we can estimate its half-life (Table 12.3).
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This method is open to criticism since in a number of cases inhibitors of protein synthesis have been shown to interfere with enzyme degradation. Thus cycloheximide stops the decline in phenylalanine ammonia lyase which follows the light induced elevation of the enzyme in potato discs and also the decline in phosphoglucomutase which occurs on slicing potato tubers. In these cases, cycloheximide is assumed to block the synthesis of the specific proteolytic enzymes necessary for the removal of the lyase and the mutase.
12.4.2—
Enzyme Inactivation
Experimentally it is difficult to distinguish between enzyme degradation and inactivation. This is reflected in the terminology, thus the enzyme described in the previous section which is thought to degrade nitrate reductase is referred to as a nitrate reductase inactivating enzyme by Wallace. A simple kinetic test enables an investigator to determine if enzyme activity is changing during the period of an assay. The rate equation for an enzyme catalysed reaction can be written:
Provided that (S ) substrate, (A ) activator and (I ) inhibitor are constant in a series of assays, the integral form of the equation is
Thus for a series of assays when the amount of product formed is plotted not against time, but against time multiplied by enzyme concentration, all the values should fall on a single curve. If the points do not fall on a single curve then it is likely that the enzyme is being degraded or inactivated in some way (Selwyn, 1964).
12.4.2.1—
Inactivation by Protein-Protein Interaction
The modulation of enzyme activity by the interaction of protein subunits is widely recognized. A number of reports suggest that interaction between different proteins may result in enzyme inactivation. For example the naturally occurring inhibitor of potato tuber invertase appears to be a protein which forms an essentially undissociable complex with invertase. The inactivation of Lemna nitrate reductase has been attributed to protein-protein interaction and ammonium adapted plants are said to contain a protein which inhibits nitrate reductase.
However, the most clearly established case is the interaction between ornithine transcarbamylase and arginase in Saccharomyces (Wiame et al., 1973). The activity of ornithine transcarbamylase was shown to decline following the addition of arginase. It was established that arginase binds to the ornithine
transcarbamylase and inhibits its activity. The binding is on a one to one basis and does not reduce the arginase activity. This interaction has not been observed in other organisms.
12.4.2.2—
Chemical Modification
A number of enzymes are inactivated by low concentrations of pyridoxal phosphate at physiological pH. The mechanism of inactivation is the formation of a Schiff base between pyridoxal phosphate and an amino group of the protein, usually the e -amino group of lysine.
Glutamate dehydrogenase and aldolase have been shown to be inactivated by pyridoxal phosphate and to be protected by their substrates, suggesting that lysine residues are involved in the active centre of these enzymes. It is difficult to assess the physiological significance of these reactions; the concentration of free pyridoxal phosphate in plants is low but the ratio pyridoxal phosphate: pyridoxamine phosphate changes with the nitrogen status of the plant and the possibility of this ratio monitoring the nitrogen status of the plant and controlling the activities of key enzyme has been discussed.
Carbamyl phosphate also reacts with e -amino groups of lysine, the rate of carbamylation varying from protein to protein. Glutamate dehydrogenase is readily inactivated by carbamyl phosphate but it is doubtful that carbamylation of enzymes has physiological significance in plants.
Enzyme catalysed chemical modification is well documented in animals and bacteria and among the important examples we note glycogen phosphorylase and glycogen synthetase of E. coli which is adenylated by ATP. Adenylation has not been observed in plants but phosphorylation, methylation and acetylation of proteins has been demonstrated. The extent to which these modifications are important regulatory mechanisms remains to be determined.
The classification of chemically modified enzymes in terms of activation or inactivation is somewhat artificial. For example phosphorylation leads to an activation of glycogen phosphorylase but an inactivation of glycogen synthetase. These chemical modifications could equally well be considered as dephosphorylation in which case glycogen phosphorylase is inactivated and glycogen synthetase activated. However, from an operational point of view, whether an enzyme is activated or inactivated is of paramount importance.
12.4.3—
Enzyme Activation
Four enzymes of the Calvin cycle and two enzymes of the Hatch-Slack pathway are light activated whilst glucose-6-phosphate dehydrogenase, an enzyme of the pentose phosphate pathway is light inactivated. It is highly probable that a number of these responses reflect a reductive process, since dithiothreitol treatment of crude extracts gives results similar to light treatment of intact seedlings. However, in some cases the reaction is certainly more complicated
than a direct response to a change in the SH/SS redox potential. A scheme for the activation/inactivation of pyruvate phosphate dikinase is shown in Fig. 12.10.
Figure 12.10
Schematic representation of the processes of activation and inactivation of
pyruvate, P: dikinase. The process leading to the irreversible inactivation
of both active and inactive enzyme in vitro apparently include an O2
-dependent oxidative reaction involving thiol groups.
(After Hatch & Slack, 1969.)
The mechanism of activation may vary from species to species. Thus the light-activation of pea NADP glyceraldehyde phosphate dehydrogenase appears to involve a conformational change produced by reduction and leads to an increase in Vmax . The spinach enzyme however, appears to need NADPH or NADP or ATP in addition to a reducing agent for activation. The activation involves dissociation of a tetramer leading to an increased affinity for NADP.
An interesting suggestion for the blue light activation of phenylalanine ammonia lyase has been proposed by Engelsma (1974). He suggests that the enzyme is present in gherkin hypocotyls in an inactive form due to inhibition by trans-hydroxycinnamic acid. Blue light (in the presence of a photoreceptor such as riboflavin) converts the trans- to the cis-hydroxycinnamic acid which is much less inhibitory. A phytochrome-mediated activation of phenylalanine ammonia lyase has also been reported in mustard cotyledons by Attridge et al., (1974). It is not clear if the cis-trans isomerization is applicable to the phytochrome-mediated activation which is inhibited by cycloheximide.
12.4.4—
Enzyme Synthesis
In the absence of activation or inactivation, the net synthesis of an enzyme is given by gross synthesis minus degradation. Net synthesis is measured as the change of activity so that gross synthesis can be measured if the rate of degradation is known. Because of the technical difficulties involved in obtaining quantitative data most workers have been concerned to develop methods which establish that the increase in activity of an enzyme is due to synthesis rather than degradation.
12.4.4.1—
Density Labelling
Gibberellic acid stimulates the synthesis of a number of enzymes in the aleurone layer of barley seeds. Filner and Varner (1967) introduced a method of demonstrating that the bulk of a -amylase which appears after treatment with gibberellic acid is due to protein synthesis. They incubated aleurone cells with H218 O so that when storage proteins were hydrolysed 18 O-labelled amino acids were formed.
When new protein is synthesized from the 18 O-labelled amino acids it will have a greater density due to the incorporation of 18 O into the peptide links. The density difference between 18 O and 16 O containing proteins allows separation by equilibrium density gradient centrifugation. The observed increase in density was consistent with the bulk of the new a -amylase being synthesized from amino acids. A major limitation is that proteolysis is required to introduce 18 O into amino acids and this restricts the method to studies of seed germination.
An alternative method is to introduce 2 H into the protein. This can be achieved by incubating a tissue in 2 H2 O so that 2 H labels the amino acids by entering at the a -carbon atom in a transaminase catlysed exchange reaction. With prolonged incubation in 2 H2 O, deuterium enters the amino acids at other points producing large changes in the density of newly formed protein.
The major advantage of the density labelling methods is that they enable the synthesis of a particular enzyme to be demonstrated without purifying the enzyme. However, the method requires expensive equipment and is time consuming. Consequently many investigators rely on data obtained by the use of inhibitors of protein synthesis. Such data is rapidly obtained and at low cost but its interpretation must always be equivocal.
12.4.4.2—
Enzyme Induction
Filner et al., (1969) have listed the numerous examples of increases in enzyme activity in response to age, hormonal or environmental changes. In many cases these increases may be due to enzyme synthesis, though the evidence is often little more than that gained by the use of inhibitors of protein synthesis. The evidence for substrate-induced enzyme synthesis is extensive in the case of nitrate reductase but in other cases, for example the induction of a specific isoenzyme of glutamate dehydrogenase, the case is not yet proven.
12.5—
Final Comments
The basic similarity of living organisms is reflected in their biochemistry, the major metabolic pathways being common to all living organisms. The diversity of living organisms is reflected in their specialized control systems. The control
mechanisms of one species may be very different from those of a closely related species. The investigator has a daunting task when he considers the amount of work necessary to establish control mechanisms over a wide range of species.
The difficulties of comprehending control mechanisms even in a single organism are no less daunting. The difficulties stem from the number of assays necessary to establish the complete rate law for a regulatory enzyme. For example the enzyme glutamine synthetase from E. coli is known to be affected by eight reactants and modifiers. Hence if 6 points per curve are necessary for a kinetic analysis, then to establish the rate law for glutamine synthetase will require in excess of 1.6 × 106 assays. The task of analysing the many enzymes involved in metabolism is frightening and when we recognize that all the enzymes interact and affect one another the system defies analysis even with modern computers.
The way ahead seems to require inspired guesses at the nature of the control mechanisms involved, followed by model building and a systems analysis—probably making linear approximations—to see if the behaviour of the biological system can be predicted.
The difficulty in analysing control systems may be compared with the difficulties in understanding economics. The main control step of glycolysis appears to be phosphofructokinase, a reaction which involves a loss of free energy of 4 Kcals per mole. This loss may be considered the cost of controlling glycolysis. Protein degradation involves a large loss of free energy and this may be the price the cell has to pay for its complicated but effective control mechanisms. The situation can be likened to the economy of surplus production in society—in the Brave New World of Aldous Huxley products were destroyed to maintain the economy—perhaps nature is a capitalist!
Further Reading
Bayer P.D. (1970) (Ed.) The Enzymes. See articles by Koshland D.E., Stadtman E.R., & Atkinson D.E. Academic Press.
Cohen G.N. (1968) The Regulation of Cell Metabolism. Holt, Rhinehart & Winston.
Davies D.D. (1973) (Ed.) Rate control of biological processes. Symposia of the Society for Experimental Biology. Vol 27.
Kun E. & Grisolia S. (Eds.) (1972) Biochemical Regulatory Mechanisms in Eukaryotic Cells. Wiley & Sons.
Milborrow B.V. (Ed.) (1973) Biosynthesis and its Control in Plants. Phytochem. Soc. Sym. Series No. 9.
Newsholme E.A. & Start C. (1973) Regulation in Metabolism. Wiley & Sons.