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.