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Allosteric enzymes may be regulated negatively or positively or both. For example, the enzyme phosphofructokinase (PFK) is a major control point for glycolysis. When there is plenty of ATP, this inhibits PFK, whereas the build-up of AMP and/or ADP signals that the cell is running low on energy and activates PFK. Thus, some allosteric enzymes have two different allosteric sites, one for an activator and the other for an inhibitor. In the case of PFK, ATP is the negative allosteric effector and AMP is the positive allosteric effector.

All allosteric enzymes consist of multiple subunits. When the allosteric effector binds, it changes the shape of the subunit to which it binds. In some cases, this also affects the assembly of the subunits (Fig. 7.41). One form of the allosteric protein exists as monomers and the other as multimers. In other cases, the subunits stay together. In this case, the shape change may be transmitted from one subunit to the next (which, as a bonus, can now bind the allosteric effector more easily). The subunits are said to undergo a concerted shape change.

Many DNA-binding proteins are also allosteric. They change their shape and their ability to bind to DNA when they bind small regulatory molecules. This allows the regulation of gene expression in response to a variety of chemical stimuli, as discussed in Chapters 9 and 10.

Enzymes May Be Controlled by Chemical Modification

Some proteins change conformation and activity after binding a small molecule. In other cases, a shape change is caused by modifying the protein chemically. Normally this occurs when a chemical group is added—one that can be removed again later. Phosphate groups are the most common examples of small molecules that affect shape change, but other groups including acetyl, methyl, and adenyl (AMP) may be effective.

allosteric protein Protein that changes shape when it binds a small molecule

A) Kinase

B) Phosphatase p

Inactive enzyme

Protein kinase

Protein kinase

Protein phosphatase

Protein phosphatase

FIGURE 7.42 Addition and Removal of Phosphate to and from Proteins

A) An inactive enzyme may be made active by the addition of a phosphate group by a protein kinase. A shape change occurs that makes the phosphorylated enzyme active. B) The active enzyme is altered back to the inactive conformation when the phosphate group is removed by a phosphatase.

The activity of many proteins is altered by adding or removing phosphate groups.

Potentially dangerous enzymes are often activated by cleavage of inactive precursors.

Phosphate groups are attached to proteins by enzymes known as protein kinases and are removed by protein phosphatases (Fig. 7.42). Control of enzyme activity by covalent modification is relatively rare in bacteria but extremely common in animal cells. About a third of all the 10,000 or so different proteins in an animal cell are phos-phorylated at any given instant. When animal cells receive signals from outside, they often respond by phosphorylating a particular set of proteins that includes both enzymes and transcription factors.

The classic example of control by phosphorylation is the synthesis and breakdown of glycogen by animal cells. Glycogen is a storage carbohydrate that is split to give glucose when cells need energy. It is made by glycogen synthase and broken down by glycogen phosphorylase (Fig. 7.43). Both enzymes are controlled by phosphorylation. Glycogen phosphorylase is active when phosphorylated, whereas glycogen synthase is inactive. As is often the case, there is a cascade of enzymatic reactions involving two protein kinases. When the cells need energy, protein kinase A phosphorylates both glycogen synthase and phosphorylase kinase, which in turn phosphorylates glycogen phosphorylase.

Some enzymes are activated by cleavage of a precursor protein to yield active enzyme. The digestive enzymes trypsin, chymotrypsin and pepsin are synthesized as longer precursors known as trypsinogen, chymotrypsinogen and pepsinogen. Only in the intestine, and safely outside the cells that made them, are they activated by cleavage of the polypeptide chain. Unlike control by binding small molecules or phosphorylation, this sort of activation is non-reversible.

Many DNA binding proteins recognize specific base sequences. Such recognition sequences are often (but not always) inverted repeats.

Binding of Proteins to DNA Occurs in Several Different Ways

A wide range of proteins binds to DNA. These proteins are involved in DNA replication, in gene expression and its control, in protection and repair of DNA, and a variety of other processes. Understanding the properties of DNA-binding proteins is of major importance in biotechnology, where they are used to control the expression of cloned genes. DNA-binding proteins are also of relevance in molecular medicine, especially in such areas as cancer and aging. Despite the great variety of DNA-binding proteins, there are some common themes in how these proteins interact with DNA.

Although some DNA-binding proteins are relatively non-specific, many recognize and bind to specific base sequences in the DNA. Almost all DNA-binding proteins fit into the major groove of DNA as this allows them to recognize and make contact with the bases. When several sites recognized by a particular DNA-binding protein are compared, they are found to have very similar, though rarely identical, sequences. Many glycogen Storage carbohydrate found both in bacteria and in the livers of animals phosphatase An enzyme that removes phosphate groups protein kinase An enzyme that adds phosphate groups to another protein

FIGURE 7.43 Control of Glycogen Synthesis and Breakdown

A four-step process is necessary to breakdown glycogen to release glucose as glucose 1-phosphate. 1) Inactive protein kinase A is activated upon binding to cyclic AMP (cAMP). 2) Activated protein kinase A uses ATP to change the inactive phosphorylase kinase to the active phosphate-bound form. 3) Activated phosphorylase kinase converts inactive glycogen phosphorylase to the active phosphorylated form. 4) Ultimately, active glycogen phosphorylase converts glycogen to glucose 1-phosphate, which is the first substrate for the process of glycolysis.

FIGURE 7.43 Control of Glycogen Synthesis and Breakdown

A four-step process is necessary to breakdown glycogen to release glucose as glucose 1-phosphate. 1) Inactive protein kinase A is activated upon binding to cyclic AMP (cAMP). 2) Activated protein kinase A uses ATP to change the inactive phosphorylase kinase to the active phosphate-bound form. 3) Activated phosphorylase kinase converts inactive glycogen phosphorylase to the active phosphorylated form. 4) Ultimately, active glycogen phosphorylase converts glycogen to glucose 1-phosphate, which is the first substrate for the process of glycolysis.

Just a few structural motifs are responsible for binding DNA in a large number of different DNA binding proteins.

DNA-binding proteins, including both regulatory proteins and enzymes that cut or modify DNA, recognize palindromes or inverted repeats in the DNA. In this case, proteins that consist of single subunits often bind to inverted repeats that are 4 to 8 bp long overall. Proteins that consist of paired subunits usually bind to inverted repeats that have two 5- or 6-base repeats separated by half a dozen bases whose sequence is relatively unimportant (Fig. 7.44).These relatively short palindromes do not form hairpins or stem and loop structures.

A vast number of different transcription factors and other regulatory proteins bind DNA by means of a relatively small number of DNA-binding domains.The best known of these motifs are the helix-turn-helix, helix-loop-helix, leucine zipper and zinc finger.

Both the helix-turn-helix (HTH) and the similar but distinct helix-loop-helix (HLH) motifs consist of two a-helices joined by a loop (Fig. 7.45).The turn or loop is shorter for the HTH motif and longer for the HLH domain. In each case, one of the a-helices fits into the major groove of the DNA double helix and makes contact with the bases. In the HTH motif, it is the second of the two helices (counting from the N-terminal end) that is responsible for DNA binding whereas in the HLH motif it is the first a-helix. Proteins with these motifs usually bind as dimers to inverted repeats in the DNA (Fig. 7.46).

The HTH domain is widely used by both prokaryotes and eukaryotes whereas the HLH motif is found mostly in eukaryotes. For example, the HTH motif is found in the Crp global activator of E. coli and in both the CI and Cro (Fig. 7.47) regulatory proteins of bacteriophage lambda. Eukaryotic transcription factors that recognize home-obox sequences and control development in multi-cellular animals use an HTH motif. [Homeobox sequences are found in the regulatory regions of genes involved in overseeing spatial and temporal development in animals (see Ch. 19).] Although the rest helix-loop-helix (HLH) One type of DNA-binding motif common in proteins helix-turn-helix (HTH) One type of DNA-binding motif common in proteins

FIGURE 7.44 Binding of Proteins to Inverted Repeats on DNA

A) Double-stranded DNA with a 5-base inverted repeat. B) A protein dimer has bound to the inverted repeat sequences on the two different strands of DNA. Note how the helical twisting of the DNA brings the two recognition sequences together and so allows the two protein subunits to bind side by side.

DNA with inverted repeats

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