Principles of Biochemistry, Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
Metabolism: An Overview
Intermediary metabolic steps can be identified using mutant organisms

An important approach to elucidating metabolic pathways involves The Study of mutant organisms that are unable to synthesize a given enzyme in its active form. Unless lethal, such a defect often results in the accumulation and excretion of the defective enzyme's substrate by the mutant. For example, several stages of Amino acid METABOLISM have been elucidated by studying inherited Metabolic Disorders in humans in which a specific enzyme is not produced (Fig. 13-18). Because such Genetic Disorders are relatively rare in humans, they cannot serve as objects of systematic study. However, they can be induced artificially in microorganisms by exposing Cells to various mutagenic agents (such as X-rays or specific chemical compounds) capable of altering The Structure of particular genes in their DNA. Mutant microorganisms obtained in this way, having lost The ability to synthesize a specific enzyme, serve as an excellent tool for studying metabolism.

Let us now examine how such mutants are utilized. Normal, non-mutant Cells of the bread mold Neurospora crassa (Fig. 13-19) can grow on a simple medium containing glucose as the sole carbon source and ammonia as the sole nitrogen source. However, when the spores of this fungus are exposed to X-ray irradiation, mutants emerge that are no longer able to grow on this simple medium, yet grow quite normally on a medium containing specific metabolites. For instance, some Neurospora mutants develop normally on a medium containing The amino acid Arginine, which is not required by non-mutant cells. Clearly, in such mutants, one of the Enzymes involved in the synthesis of arginine from ammonia is either inactive or not produced at all. Because of this arginine deficiency, the mutant cells are unable to synthesize Proteins containing arginine and therefore fail to grow. However, they will grow normally if arginine is added to the nutrient medium along with glucose and ammonia. Such mutants with a disruption in a biosynthetic pathway, whose growth can be restored by supplying them with the normal product of that process, are called auxotrophic mutants (from the Greek "aukso", meaning to increase, referring to the elevated Nutritional Requirements of these mutants).

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Fig. 13-18 Cases of inherited abnormalities in phenylalanine metabolism observed in humans. Each of these cases is associated with the loss of function of a single Gene. The study of these disorders has made it possible to establish The Nature of the intermediate products of phenylalanine metabolism.

Fig. 13-19. The vegetative form, or mycelium, of the bread mold Neurospora crassa. Mutant strains of this Organism are easily obtained and have proven to be extremely useful tools in the study of Various metabolic pathways. Experiments with Neurospora mutants led to the formulation of the "one gene-one enzyme" hypothesis.

Not all Neurospora mutants that have lost the ability to synthesize arginine are identical; they differ depending on which stage of arginine Biosynthesis is impaired (Fig. 13-20). A set of different arginine-requiring mutants can be used to identify the intermediate stages in The sequence of enzymatic reactions that comprise the synthesis of this amino acid. If mutant I (Fig. 13-20) is grown in a medium with a very small (limiting) amount of arginine, the cells grow until all available arginine is consumed for Protein Synthesis. At the same time, precursor D accumulates in the culture medium because it cannot be converted into arginine, as mutant I has a block in the specific enzymatic reaction responsible for this conversion. If we now remove the cells of mutant I from the culture medium by filtration and introduce cells of mutant II—which also requires arginine, but for a different reason, namely the inability to synthesize intermediate product D—the culture medium from mutant I will evidently support the growth of mutant II because precursor D is present in it. However, the filtered culture medium of mutant II should not support the growth of mutant I. We can therefore identify the precursor accumulating in mutant I by the growth rate of mutant II. Ultimately, it was precisely through this approach that all four precursors of arginine—A, B, C, and D—were identified. Such cross-feeding experiments using auxotrophic mutants of Neurospora crassa or Escherichia coli have elucidated the biosynthetic pathways of many Amino Acids.

Fig. 13-20. Auxotrophic mutants of Neurospora crassa that have lost the ability to synthesize one of the enzymes (indicated in the figure by a red bar) involved in The biosynthesis of arginine (Arg) from precursor A As a result of mutation. Substances B, C, and D act as intermediates in this conversion. Mutant I lacks enzyme E₄, but it can be grown on a medium enriched with arginine. Under these conditions, intermediate product D accumulates in the culture medium. Mutant II lacks enzyme E₃, but retains The activity of E₄. Therefore, it can grow if either arginine or intermediate product D, produced by mutant I, is added to the medium. Similarly, mutant III, lacking enzyme E₂, grows on a medium containing C, D, or arginine, since it can convert intermediates C or D into arginine.