ALA synthesis

Gabaculine (3-amino-2,3-dihydrobenzoic acid) is a potent mechanism-based inhibitor of certain pyridoxal phosphate-linked enzymes. In plants, it is a potent inhibitor of chlorophyll biosynthesis the inhibition can be reversed by Gabaculine and another transaminase 

Leaves of gabaculine-treated plants appear pale yellow, and pigment analysis shows a specific effect only on chlorophyll and phytochrome. Detailed analysis of photosynthetic pigments in gabaculine-treated plants shows a greater effect on chlorophyll biosynthesis in leaves which develop after treatment (Table 5.1). Leaf expansion continues in the absence of chlorophyll biosynthesis, and after seven days, once expansion is complete. 

Knowledge of the inhibition of ALA synthesis by gabaculine provoked Flint to attempt to design some further inhibitors of ALA synthesis and evaluate their potential as herbicides. Some proved to be potent inhibitors and active herbicides. To date, no commercialization of ALA synthesis inhibitors has occurred, but these inhibitors have been successfully used to probe and gain information on the biosynthesis of ALA and tetrapyrroles in plants. 

Gabaculine-treated chlorophyll-deficient leaves respire normally, and there is normal cytochrome c oxidase in isolated mitochondria, providing evidence for the C5 pathway in plastids and the Shemin pathway in mitochondria and indicating the specific effect of gabaculine on the plastid 

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In plants, algae and many bacteria there is an alternative route for ALA synthesis that involves the conversion of the intact five-carbon skeleton of glutamate in a series of three steps to yield ALA. In all organisms, two molecules of ALA then condense to form porphobilinogen in a reaction catalyzed by ALA dehydratase (also called porphobilinogen synthase) (Fig. 2a). Inhibition of this enzyme by lead is one of the major manifestations of acute lead poisoning. 

Figure 1 An example of the way metallo-enzymes are under controlled formation through both controlled uptake (rejection) of a metal ion and controlled synthesis of all the proteins connected to its metabolism and functions. The example is that of iron. Iron is taken up via a molecular carrier by bacteria but by a carrier protein, transferrin, in higher organisms. Pumps transfer either free iron or transferrin into the cell where Fe + ions are reduced to Fe + ions. The Fe + ions form heme, aided by cobalamin (cobalt 2 controls) and a zinc enzyme for a-laevulinic acid (ALA) synthesis. Heme or free iron then goes into several metallo-enzymes. Free Fe + also forms a metallo-protein transcription factor, which sees to it that synthesis of all iron carriers, storage systems, metallo-proteins, and metallo-enzymes are in fixed amounts (homeostasis). There are also iron metallo-enzymes for protection including Fe SOD (superoxide dismutase). Adenosine triphosphate (ATP) and H+ gradients supply energy for all processes. See References 1 -3.

Note these results suggest that ALA synthesis in plants may occur by a completely different pathway from that in animals (where glycine condenses with succinyl-S-CoA to form ALA directly). 

The reaction mechanism consists of formation of a Schiff base by pyridoxal phosphate with a reactive amino group of the enzyme entry of glycine and formation of an enzyme-pyridoxal phosphate-glycine-Schiff base complex loss of a proton from the a carbon of glycine with the generation of a carbanion condensation of the carbanion with succinyl-CoA to yield an enzyme-bound intermediate (a-amino-yS-ketoadipic acid) decarboxylation of this intermediate to ALA and liberation of the bound ALA by hydrolysis. ALA synthesis does not occur in mature erythrocytes. 

Figure 1-2 ALA synthesis by ALAS or the C5 pathway. The enzymes of the C5 pathway and their cofactors are as follows (a) glutamyl-tRNA reductase (requires Mg-ATP) (b) glutamyl-tRNA reductase (pyridoxal 5 -phosphate dependent and requires NAD(P)H) and (c) glutamate 1-semialdehyde aminotransferase.

The first product formed by condensation of glycine and succinyl CoA is 5-aminolaevulinic acid or ALA. This reaction is catalysed by the enzyme ALA synthase and requires the cofactor, p)rridoxyl phosphate. ALA synthesis occurs entirely within the matrix of the mitochondrion and is very tightly regulated by control of the enzyme that makes it, control of the transport of the enzyme from the cytosol into the mitochondrion, and finally by inhibition of ALA synthase by the final product, heme. 

AMINOLEVUUNATE (ALA) SYNTHESIS IN SYNECHOCOCCUS 301 REQUIREMENT FOR tRNA " AND PYRIDOXAMINE PHOSPHATE. 

Fig 2 Reconstitution assay for ALA-synthesis in vitro using HPLC analysis of C-labelled products ... 

The soluble RNA fraction, which was prepared from the Chlorophyllin-Sepharose bound material was separated by HPLC as described (7). Only one tRNA could be detected using the ligase from the Blue-Sepharose fraction (Fig. 3). This tRNA could be used to reconstitute ALA-synthesis. The same result has been described for barley (3) whereas Methanobacterium thermoautotrophicum (19) and Scenedesmus obliquus (16) possess two different tRNAs which can both be used to reconstitute ALA synthesis. 

Aminolevulinate (ALA) Synthesis in Synechococcus 6301 Requirement for IRNA and Pyridoxamine Phosphate 807... 

Approximately 210 mg of heme are formed daily in the bone marrow of the adult to replace the hemoglobin lost through red cell breakdown. Since eight molecules of ALA are required to form one molecule of heme, about 358 mg of ALA are required for this amount of heme synthesis. In the inherited disease AIP, the liver may readily produce this much or more ALA yet normally the liver makes only about 15% of the ALA that is made by the bone marrow. It is obvious, therefore, that in the liver there is an important control mechanism for ALA synthesis revealed by this disease. Depending on the type of hepatic porphyria, the ALA which is produced may be excreted together with porphobilinogen in the urine, or it may excreted in the form of porphyrins in the urine and feces [2,4,6,11]. 

The enzyme from Rhodopseudomoms spheroides has maximal activity [38] at pH 6.9. The K for pyridoxal phosphate is 5 x 10 M, for succinyl-CoA 2.2 x 10" M, and for glycine 3 x 10" M. Inhibitors of this enzyme reveal that SH groups are required for activity. In erythrocytes a-KG is a competitive inhibitor of glycine utilization concentration of a-KG above 1 mM inhibits ALA synthesis. 

There still remains to be explained the fact found by Vogel et al. [43] that iron-deficient bird erythrocytes have a diminished rate of ALA synthesis. When such erythrocytes are incubated for 30 minutes with Fe , the rate of ALA synthesis increases two- to three-fold. Neither hemolysates nor cell particles from iron-deficient cells respond to the addition of iron. 

Another reaction to form ALA is by way of the transmination of L-alanine with y, 5-dioxovalerate [Neuberger and Turner, 44]. The reaction toward ALA formation is favored. The reaction is dependent on pyrixodal phosphate and requires free thiol groups. The enzyme has l5een partially purified from Rhodopseudomonas spheroides. Its significance for ALA synthesis in vivo is not known. Other precursors have been hypothesized by Tait [12]. 

Synthesis of succinyl-CoA in mammalian cells such as the red cell and liver cell can be accomplished either from a-KG or from succinate. The formation of succinyl-CoA occurs in the mitochondria as part of the citric acid cycle reactions. The requirement for a citric acid cycle to form ALA or protoporphyrin or heme has been shown by tracer studies with acetate and succinate [39], and by inhibition studies with malonate, Ira j -aconitate, fluoracetate, and arsenite [49]. The requirement for an electron transfer system from the citric acid cycle to O2 has been shown by inhibition studies with anaerobiosis and CO. The requirement for oxidative phosphorylation has been shown by dinitrophenol inhibition of ALA synthesis dinitrophenol may also inhibit ALA-synthetase [3,49]. 

The limiting enzyme for CHL synthesis is probably the one that brings about the synthesis of ALA as shown by the following experiments. Illumination of the dark-grown leaves formed CHL, but inhibitors of protein synthesis prevented CHL formation (but not if ALA was supplied). The lifetime of ALA-synthetase was found to be less than 1.5 hours as determined by the use of inhibitors of protein synthesis. When dark-grown leaves were fed various possible precursors of ALA and illuminated, CHL synthesis was not enhanced over that of controls this result suggested that these substrates were not limiting ALA synthesis. 

The synthesis of the enzyme that makes ALA was not blocked by actinomycin D or other inhibitors of RNA synthesis. This result suggests that there may be suflicient of various RNA compounds already stored in the proplastid to be nonlimiting during the first 6 to 12 hours of illumination to serve for the synthesis of ALA-synthetase. If inhibitors of RNA synthesis are efiective in these cells, then the effect of illumination may be to bring about the production of the limiting enzyme for ALA synthesis by activating the translation rather than at the transeription step. A summary of the hypotheses on the control mechanism for CHL synthesis is presented in Fig. 11. 

Figure 5.5. Proposed biosynthetic sequence of ALA synthesis from glutamate to the condensation of two ALA molecules, forming porphobilinogen. The enzymes catalyzing the steps in the pathway are (1) glutamyl-tRNA synthetase, (2) glutamyl-tRNA dehydrogenase, (3) ALA synthase (GSA aminotransferase), and (4) ALA dehydratase.

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