Amino acid channelling

The organization of multienzyme complexes can result in compartmentalization or channelling of specific metabolites from one enzyme to another without equilibration with other pools of that metabolite. Channelling of metabolites is important for many metabolic pathways, including fatty acid synthesis, glycolysis and the urea cycle (Srere, 1987; Watford, 1989; Srere and Ovadi, 1990). Channelling has the potential of increasing the efficiency of a metabolic pathway by limiting loss of substrate. Additionally, the transport of substrates across cell membranes often depends on the coupling of an enzyme of that metabolite's utilization to the transport protein. Consequently, the channelling of substrates through complex metabolic pathways often begins at the cell membrane. One prerequisite for channelling is the existence of a structural organization for the components of the pathway that can lead to the catalysis of sequential reactions without the dissociation of intermediates. A second prerequisite for channelling is the demonstration of distinct pools of substrate and intermediates that do not freely mix with the respective general intracellular pools. Evidence supporting these two prerequisites implicates the channelling of amino acids arising from proteolysis towards protein synthesis.

The structural organization that implicates the channelling of aminoacyl-tRNA to protein synthesis is well documented. Aminoacyl-tRNA synthetases are enzymes that activate amino acids and esterify them to tRNAs. Many of the aminoacyl-tRNA synthetases exist in a multienzyme complex. Presently, ten of the 21 synthetase activities have been isolated from the multienzyme complex (isoleucine, leucine, lysine, methionine, arginine, proline, phenylalanine, gluta-mine, glutamate, aspartate) and it is suspected that additional enzymes are lost in the purification procedure (Schimmel, 1987; Yang and Jacobo-Molina, 1990). The enzyme complex has been identified in many cell lines as well as myoblasts (Shi et al., 1991). The synthetase complex is physically associated with elongation factor eEFl (Sarisky and Yang, 1991), one of the primary regulators of rate of protein translation. Several of the enzymes in this multienzyme complex have amino terminal hydrophobic regions that associate with lipids, presumably in one of the cell membranes (Huang and Deutscher, 1991). With gentle homogenization of chick embryos, aminoacyl-tRNA synthetases purify in the microsomal fraction. Although it has not been shown which membrane the complex is associated with, the linkage between tRNA charging enzymes and amino acid transport across membranes is implicated. Quay et al. (1975) have shown that the transport of leucine is linked to and regulated by leucyl-tRNA synthetase activity.

Distinct substrate pools have also been identified. Sivaram and Deutscher (1990) provided evidence for two pools of arginyl-tRNA, one which is free in the cytosol and is involved in the post-translational modification of proteins, and a second which is a component of the aminoacyl-tRNA synthetase complex. Further, neither free arginyl-tRNA nor free phenylalanyl-tRNA are used for ribosomal protein synthesis, but the tRNAs formed in the multienzyme complex are efficiently used (Negrutskii and Deutscher, 1991). This suggests that aminoacyl-tRNAs formed in the multienzyme complex are transferred directly from the synthetase to the elongation factor and the ribosome without mixing with the total fluid of the cell, representing the channelling of aminoacyl-tRNA for protein synthesis.

As previously described, distinct substrate pools of free amino acids are apparently used for the charging of tRNA. This pool is separate from the total intracellular pool of free amino acids. The extent and time course of dilution of the specific activity of the radiotracer by unlabelled free amino acids in the cell or from proteolysis, as reflected in aminoacyl-tRNA, indicates the source of amino acids used for protein synthesis. Further work is needed to determine the extent of this amino acid partitioning and the method of its regulation.

Putative charging of tRNA from amino acids channelled directly from protein degradation is understandable both energetically and nutritionally. By directly reutilizing a significant proportion of the amino acids released from protein degradation, the cell can prevent the efflux of essential amino acids from the cell and minimize their loss to catabolic pathways. Efficient reutilization of amino acids could result in decreased energy requirements associated with their transport into the cell. Additionally, efficient channelling of amino acids arising from the diet or other tissues across the cell membrane directly to the aminoacyl-tRNA synthetases would preclude the possibility of loss of the amino acid to oxidation.

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