Chapter 3:  Protein Function and Malfunction in Cells

Active sites

Enzymes are biological catalysts that speed up chemical reactions that otherwise would occur too slowly for cells to function. The substrate molecules fit into an enzyme’s active site. Enzymes do their job of catalysis by physically grappling with the substrate molecules, interacting with them to make or break chemical bonds. Figure 3-23 diagrams the general concept and shows two proposed modes of enzyme action. Enzymes are highly specific for the chemical reactions they catalyze, and the specificity lies in the precise fit between substrate and active site. Figure 3-24 shows the active site of the enzyme carboxypeptidase, a digestive enzyme that cleaves polypeptides of food proteins between the amino acids glycine and tyrosine. As it binds its substrate (Figure 3-24a), it undergoes a conformational shift that promotes catalysis.

Much of the globular structure of an enzyme is nonreactive material that simply supports the active site. However, the precise shape and binding properties of the active site are crucial for proper enzyme function, and a set of highly specific amino acids forms the lining of the active site. The codons for these amino acids tend to be clustered in the gene, but because of polypeptide folding some codons might be outside the cluster. In the following diagram the codons of the active site of a hypothetical protein are shown in white; the remaining codons of the protein, in black:

Enzyme pathways

Cells synthesize essential cellular substances step-by-step from compounds in the environment. This is done in a series of enzyme-mediated reactions called biochemical pathways. For example, the haploid fungus Neurospora synthesizes its own arginine (an amino acid) in a series of sequential biochemical steps. The immediate precursors in the pathway are ornithine and citrulline, as shown in the accompanying diagram. Ornithine is made from earlier precursors not shown; then it is converted into citrulline, which in turn is converted into arginine. These conversions are catalyzed by enzymes: we can label them enzymes 1 through 3, coded by three different arginine-synthesizing genes that we can designate genes 1 through 3:

Hundreds of biochemical pathways involving thousands of reactions take place inside living cells. Since most of these reactions are catalyzed by enzymes, we see a glimmer of the pivotal position that genes play in controlling the chemistry of life.

Mutations and a short course in nomenclature

In genetics the standard organismal phenotype is called the wild type, because this is the type observed in the wild, in other words, in nature. All essential genes must be capable of producing their functional products in order to produce this wild-type phenotype. These normally functioning alleles are called wild-type alleles.

Mutations, changes in the DNA sequence of a gene, occur spontaneously in nature. Since alleles are forms of genes, mutations by definition create new alleles. In genetic research, a gene is generally symbolized by a letter or several letters based on the word describing the phenotype produced by a mutant allele. Then the corresponding wild-type allele is designated by the addition of a superscript plus sign (+). For example the wild-type eye color of the fruit fly Drosophila is red, but a mutant allele of a gene on the X chromosome produces white eyes, so the mutant allele is designated w and its wild-type allele is w⁺.

As we saw earlier, the haploid fungus Neurospora, like most microbes, can synthesize all essential cellular compounds (such as amino acids) from simple inorganic substances in the growth medium. Therefore these organisms are said to be prototrophs (“self-feeders”). Some mutations render the fungus incapable of making specific substances. A mutant organism thus formed is called an auxotroph (“outside feeder”) because it must obtain some essential compound from the environment. Although the mutations would be lethal in nature, in the laboratory the mutant strains can be kept alive by supplying the missing substance in the growth medium. For example, some mutations result in the inability of a strain to make its own arginine, the synthesis of which was described in the previous section. A mutant allele is designated arg (standing for “arginine-requiring”), and its wild-type counterpart, which confers ability to make arginine, is arg⁺. Sometimes the + symbol is used by itself when the meaning is clear, so in the Neurospora example the two alleles would become arg and +, with the tacit understanding that the + corresponds to the wild-type allele of that particular gene.

An arg auxotroph will grow if arginine is added to the growth medium. Recall that the final portion of the arginine biosynthetic pathway is

so an auxotrophic arg mutation could be in any one of the three genes controlling these conversions. The three possibilities can be distinguished because a mutant blocked in the last step (controlled by gene 3) would grow only if supplemented with arginine; a mutant blocked in the middle step (gene 2), if supplemented by arginine or citrulline; and a mutant blocked in the first step (gene 1), by arginine, citrulline, or ornithine. This logic was used in the original elucidation of this pathway, as described in Genetics in Process 3-1. We could call the three genes (using their wild-type alleles) arg-1⁺, arg-2⁺, and arg-3⁺.

In some situations geneticists use upper- and lowercase letters to designate alleles, such as A and a. In this symbolism there is no designation of wild type. This is appropriate in cases in which the wild-type allele is not known, for example, in extensively interbred lines of plants or animals. It is also useful in naming alleles that determine the morphs of a polymorphism because all the morphs are wild-type.

Types of mutations

The simplest mutant alleles show altered DNA sequence at only one position in the gene. Such a mutant allele is composed of normal nucleotide sequence except in the area of change, known as the mutant site. Because of the way the genetic code is read during translation, a mutation that changes one base pair for another is often found to be a change of exactly one amino acid. Because of colinearity of gene and protein primary structure, mutations in the nucleotide sequence in DNA change the amino acid chain in precisely the same relative position (see Genetics in Process 3-2). A change of a single nucleotide in a codon at a crucial functional site in the protein (such as the active site of an enzyme) can result in an amino acid replacement leading to aberrant function. For example, a C·G → T·A nucleotide pair change can result in the replacement of proline by leucine, an amino acid with quite different properties that could have adverse effects on protein function.

Another common type of simple mutation is addition or deletion of single nucleotide pairs. These are called frameshift mutations because they cause the proper reading of the message to be off by one in every amino acid translationally “downstream” from the mutant site. For example, consider the effect of a deletion of a nucleotide on the following mRNA segment:

All the downstream codons will be translated incorrectly, and most of the corresponding amino acids will be incompatible with protein function.

Effects of mutations on cellular function

Generally, mutations result in reduced protein function or no protein function. A mutation with reduced function is called a leaky mutation because some of the wild-type function “leaks” through into the phenotype. A mutation that results in no protein function is called a null mutation. Changes that do not affect the function of a protein are called silent mutations.

Some mutations alter the information-transfer process rather than directly altering the genetic code. For example, some mutations produce malfunction not through any effect on amino acid sequence but on intron splicing. Since intron splicing depends on specific nucleotide sequences at the exon-intron boundaries and inside the intron, if these sites are mutated, the intron cannot be excised and no functional mRNA will be produced. A similar mutation alters the regulation of the gene. For example, the promoter sequence to which the RNA polymerase binds is crucial, and if changes occur in this sequence, the gene might not be transcribed at all or might be transcribed at abnormally low (or high) rates. Finally, mutations in the untranslated 3′ tail of the transcript can have marked effects on mRNA stability.

Some examples of mutant sites, together with their phenotypic outcomes, are shown in Figure 3-25 on page 74.

Genetic disease

A common cause of genetic disease in humans is enzyme deficiency caused by mutation. Human cells carry two chromosome sets, so all genes are represented twice (gene pairs). Normally both are wild-type alleles. However, individuals carrying a pair of defective alleles of a gene coding for an enzyme will show reduced or no enzyme activity and express disease symptoms. Table 3-4 gives some representative examples and suggests the magnitude of genetic involvement in human disease. Figure 3-26 on page 76 shows a corner of the human metabolic map to illustrate how a set of diseases, some of them familiar to us, can stem from the blockage of adjacent steps in biosynthetic pathways. Consider the disease phenylketonuria (PKU), shown in part of Figure 3-26. A defect in the enzyme phenylalanine hydroxylase causes a buildup of phenylalanine (originating from dietary protein). At high concentrations phenylalanine is converted into phenylpyruvic acid, a substance that interferes with the development of the nervous system, giving rise to mental retardation in an infant with two copies of the defective allele. If the high level of phenylpyruvic acid is detected soon after birth, the baby can be placed on a special low-phenylalanine diet and develops without retardation.

Let us return to cystic fibrosis, the disease introduced at the beginning of the chapter. This is not caused by an enzyme deficiency but by inactivity of a protein that controls the passage of chloride ions through membranes in secretory tissue (Figure 3-27). The most commonly found mutation is a deletion of three nucleotides, which should result in the removal of the amino acid phenylalanine from the primary sequence of the protein, as shown in Figure 3-28. The position of the expected deletion in the protein is shown in Figure 3-27. However, in patients with cystic fibrosis, defective proteins lacking the amino acid phenylalanine at the position of the deletion are not found at all in the membrane; there seems to be a way for the cell to detect and destroy the defective protein. Nevertheless, the lack of the channel protein and the resultant upset of the chloride and sodium balance leads to the thick mucus that causes the respiratory symptoms and to other problems.