Enzymes of cinnamic acid metabolism in plants

The wide variety of phenolic compounds found in higher plants originate from cinnamic acid. Derivatives of this molecule are formed by two types of conversion: hydroxylation of the benzene ring, and activation of the side-chain. The first hydroxylation is para to the side-chain and gives rise to p-coumaric acid. The necessity of a carboxylic activation step prior to side-chain conversions which lead to complex phenolic compounds by reduction, esterification, andbeta;-oxidation or condensation, is suggested by thermodynamic considerations, and by analogy with simpler reactions which occur in higher plants.
In this thesis the extraction of two enzymes from higher plants is described: one catalysed the para-hydroxylation of cinnamic acid, and the other the activation of cinnamic acid. Damage to the enzymes by the high concentrations of phenols and oxidising enzymes released during extraction was reduced by the use of acetone powders, the inclusion of antioxidants such as ascorbate in the extraction buffers, and the use of insoluble polyvinylpyrrolidone as a phenolic adsorbent.
The activating enzyme
The work on the activating enzyme was concerned with the extraction, measurement, and characterization of an enzyme capable of catalysing the activation of cinnamic acid, distinct from the enzyme catalysing the activation of acetate.
Four assays for the activating enzyme, based on the characteristics of a CoA-dependent activation proceeding through an acyl-adenylate intermediate, were developed: the hydroxamate test for activated carboxylic groups in which hydroxylamine reacts with an activated intermediate to give the hydroxamic acid, detectable either spectrophotometrically by its ability to form a purple complex with ferric ions under acidic conditions, or chromatographically, using [2-14C] cinnamic acid as substrate; the measurement of the appearance of cinnamyl-CoA, which has a specific absorption maximum at 311nm.; the measurement of cinnamate-dependent pyrophosphate exchange, which may occur when [32P] pyrophosphate is added to the reaction system in the absence of CoA; and an estimation of the disappearance of the sulphydryl group of CoA, measured spectrophotometrically by the nitroprusside reaction.
Aqueous extracts of acetone powders, and preparations made by extraction of plant material in buffers containing ascorbate, when assayed by the spectrophotometric hydroxamate assay, showed very high activity towards acetate and other aliphatic acids, but little or no activity towards cinnamate. There was some indication that ammonium sulphate fractionation gave partial separation of acetate- and cinnamate-activating systems. The two activities also displayed a different pH-dependence and a different stability. They were widely distributed in plant tissues.
With enzyme preparations from leaves of spinach beet plants grown outside in a hot and dry summer, and extracted in the presence of polyvinylpyrrolidone, exceptionally high levels of cinnamate-activating enzyme were measured by the spectrophotometric hydroxamate assay. The activation was very sensitive to the presence of air, and to the presence and type of thiol reagents. Maximum activation occurred ion the presence of mercaptoethanol and under an atmosphere of nitrogen. In contrast, the activation of acetate was unaffected by air and by thiol reagents.
After this successful series of experiments it was found impossible to repeat the extraction of high levels of cinnamate-activating enzyme from leaves of plants grown under a variety of conditions. The two sensitive radiochemical assays, and the assay for cinnamyl-CoA were therefore developed, and all plant material was grown under controlled conditions, in order to define its physiological state more exactly. Extracts low in mitochondrial content were prepared in the presence of sucrose to reduce the level of ATP-ase contamination.
The pyrophosphate exchange method measured cinnamate-dependent exchange catalysed by extracts of spinach beet leaves in a few cases. This exchange was completely inhibited by Co, as in reports of other substrate-dependent exchanges catalysed by CoA-dependent enzymes.
The formation of [14C] cinnamyl-hydroxamate was catalysed by extracts prepared from a variety of plant leaves (spinach beet, Beta vulgaris; spinach, Spinacia oleracea; Runner bean, Phaseolus vulgaris, and pea, Pisum sativum). The reaction appeared to be partially inhibited by p-coumaric acid, but not by other phenolic acids. There was a clear relationship between the age of a spinach beet seedling, and the activity extracted from its leaves. The development of the cinnamate-activating enzyme was inversely related to the level of betalain pigment in the leaf, and it was greater when the plant was grown under conditions of high illumination, containing a relatively high proportion of red light.
The cinnamyl-CoA assay showed activation with many of the extracts active with the [14C] cinnamyl-hydroxamate method. It also demonstrated that the activating enzyme was not reactive towards the substituted cinnamic acids tested, that the reaction had a broad pH optimum between 7.5 and 8.0, and that the Km value for cinnamate was 4.25 andtimes; 10andminus;3M. This assay measured the greatest activity in enzyme preparations.
Comparison between the results obtained with the different assays is difficult because they measured the time-course and extent of activation differently. Nevertheless certain conclusions can be drawn. The preliminary studies showed clearly that the cinnamate-activating system is distinct from the acetate-activating system. The more sensitive assays showed that the cinnamate-activating system is dependent on CoA, and may be specific for cinnamic acid, although p-coumaric acid also may be activated. The enzyme probably enjoys a wide distribution in higher plants but its presence is dependent on the physiological state of the plant, and may be under light control.
Different assay methods measured maximum activation at different pH values, and measured different affinities of the enzyme for cinnamic acid. The pH optimum and the Km values measured by the cinnamyl-CoA assay are expected to give the true values of these parameters, since this assay measures the overall reaction in the absence of hydroxylamine, or excessive concentrations of pyrophosphate, and in the presence of CoA, which may be necessary for the full activity of the enzyme. It is also the only assay which measures an initial rate.
The hydroxylating enzyme
The initial studies on cinnamic acid hydroxylase were leaf disc experiments, in which formation of labelled p-coumaric acid from exogenous [2-14C] cinnamic avid was demonstrated in material from spinach and spinach beet.
Preliminary studies on crude extracts of cinnamic hydroxylase from the leaves of plants in which the hydroxylation has already been established by leaf disc studies, showed that little activity was measured by two different spectrophotometric assays for phenols. A method using [2-14C] cinnamic acid as substrate estimated [14
C] p-coumaric acid after chromatographic separation from the substrate. The formation of p-coumaric acid was dependent on NADPH or NADH and tetrahydrofolic acid. The preference for NADPH or NADH was dependent on the type of plant extract, but no absolute specificity was found.
Control values obtained with boiled extracts always gave greater radioactivity in p-coumaric acid than test values with untreated enzyme, even under optimum conditions of incubation. No completely adequate explanation for this can be offered, although some artifact in the experimental procedure is suspected, since a non-enzymic hydroxylation is unlikely. The activity which may be present, considering this limitation, is similar to the cinnamic hydroxylase activity described in several other tissues.