Lecture 21 Summary

There are numerous ways in which the activity of enzymes are regulated. More generally, the regulation of protein function by specific ligand interactions (including interactions with other proteins) and covalent modifications are key features in the organizational and control mechanisms of living organisms. The regulatory strategies we will consider are the following:

1. Allosteric control (Examples: hemoglobin and ATCase)

2. Multiple forms of enzymes ("isozymes")

3. Reversible covalent modification (Primary example: phosphorylation and dephosphorylation by kinases and phosphatases, respectively)

4. Proteolytic activation (Examples: processing of proenzyme forms of digestive enzymes; blood clotting cascade)

Covalent modification as a regulatory mechanism

Covalent modifications of proteins - the attachment of a wide range functional groups or molecules via covalent bonds - play a prominent role in the functional regulation of proteins, which in turn can have significant effects on metabolic pathways, cellular properties and fates, and the control and integration of processes of whole organisms. A number of cancer causing oncogenes are corrupted forms of enzymes known as protein kinases that carry out covalent modifications of other proteins by the transfer of phosphate groups to certain residues of target proteins. Such kinases normally function in the proper control of cellular growth, proliferation, and differentiation

Here we briefly review the variety of covalent modifications of proteins that have regulatory effects. Covalent modifications include attachment of small groups such as phosphate and acetyl, to larger species such as lipid and carbohydrates, and even other proteins, as in ubiquitination. (We consider specific proteolytic cleavage events as a sepaarte category of regulatory mechanism since although it is, strictly speaking, a covalent modification in that peptide bonds are broken, it is an irreversible process). These modifications can activate, attenuate, or terminate protein activity. We then have a closer look at phosphorylation of proteins as a regulatory mechanism. As noted above, phosphorylation of proteins is carried out by enzymes called protein kinases. Phosphorylation is reversible, and dephosphorylation is accomplished by protein phosphatases, enzymes that hydrolyze phosphate groups attached to proteins.

cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA)

We have seen how the activity of allosteric proteins such as hemoglobin and ATCase can be regulated by binding of small molecule ligands. The activity of protein kinases can be similarly regulated and we consider here a particularly instructive and important example. The protein kinase known as protein kinase A (PKA), or cAMP-dependent protein kinase (cAPK), is regulated by 3',5'-cyclic AMP (cAMP). cAMP is the classic example of what is referred to as a "second messenger", a molecule that is produced by a primary signaling event. Typically, the primary signal the binding of a hormone to an extracellular domain of an integral membrane protein that functions as a specific receptor for the hormone. In this case, binding of a hormone such as epinephrine or glucagon set in motion a chain of events that stimulate production of cAMP within the cell. The binding of cAMP to regulatory subunits of PKA activates it by an allosteric mechanism. The activated kinase then phosphorylates a variety of cellular targets, altering their activities and thereby causing specific physiological responses

Isoforms

Isoforms are distinct but closely related forms of a protein, typically encoded by different genes. In other words, they are paralogs that have not undergone significant functional divergence. Although they share the same basic function, isoforms differ in properties such as tissue distribution or expression levels that vary according to developmental stage. An example is the γ globin gene that is expessed during fetal development, which replaces β globin chains of adult hemoglobin (HbA) to form the higher-affinity fetal hemoglobin (HbF). This highlights the regulatory strategy made possible by the existence of isoforms. The isoforms can differ in allosteric properties, or ligand-binding affinities. Isoforms of enzymes are referred to as isozymes, and these can differ in their kinetic parameters, range of substrate specificities and preferences, and allosteric properties.

An example of isozymes (discussed in BTS6, Ch.10, p.283) are the H and M isozymes of lactate dehydrogenase (LDH) [EC 1.1.1.27], a tetrameric enzyme interconverts pyruvate and lactate. The forward conversion is important for maintenance of redox balance under anaerobic conditions since it is accompanied by the regeneration of NAD⁺ from NADH. The reverse conversion allows the continuation of high energy-yielding oxidative metabolism. The H form is predominent in heart muscle, which operates aerobically. The M form is expressed in skeletal muscle, which operates occasionally under anaerobic conditions, when exertion levels consume energy at a rate that exceeds the rate at which oxygen can be transported to the tissue to maintain oxidative energy metabolism. Under these conditions, skeletal muscle produces lactate from pyruvate, and exports the lactate to the bloodstream where it is circulated to other tissues - such as liver and heart muscle - that take up lactate and metabolize it by re-oxidizing it back to pyruvate.

The M₄ version of LDH excels at converting pyruvate into lactate, ideal for skeletal muscle performing anaerobic exercise. This form is not inhibited allosterically by high levels of pyruvate. On the other hand, the H₄ version of LDH has higher substrate affinity, but is subject to allosteric inhibition by pyruvate (the product of the LDH reaction under the aerobic conditions prevailing in heart muscle). Thus, the H₄ LDH is better adapted to the aerobic utilization of lactate as an energy source. The two isoforms of LDH monomers can be combined into heterotetrameric species with intermediate properties, allowing for fine-tuning of LDH activity to suit tissue- and developmental stage-specific needs.

Learning objectives

• List and provide examples of each of the four regulatory strategies discussed..
• Define kinase and protein kinase. Name the common residue targets of protein kinases.
• Describe the how phosphorylation of protein targets alters their biochemical properties.
• Describe the signal transduction pathway based on intracellular cyclic AMP.
• Describe the structural, catalytic, and regulatory properties of protein kinase A.

Page update in progress, 11-01-09

References

1. Zheng, J., et al. (1993). Acta Crystallogr., D49: 362-365.
2. Lactate dehydrogenase - Molecule of the Month @ Protein Data Bank