Lecture 22 Summary

Introduction to regulatory strategies. Allosteric regulation of ATCase

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 aspartate transcarbamoylase (ATCase).

2. Multiple forms of enzymes ("isozymes")

3. Reversible covalent modification (Primary example: phosphorylation and dephosphorylation by kinases and phosphatases, respectively. The text discusses the example of glycogen phosphorylase, which is activated by phosphorylation)

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

Drug design

Historically, pharmacology has relied on empirical observations. More recently, as knowledge about biological function at the molecular level has accumulated, it has fostered attempts to exploit understanding of molecular mechanisms in development of therapeutic interventions. (12: 394-400)

ATCase: "the hemoglobin of enzymology"

Aspartate transcarbamoylase [ATCase, EC 2.1.3.2], which catalyzes the committed step in the pathway for pyrimidine nucleotide synthesis in bacteria, is dubbed "the hemoglobin of enzymology" since it is the most well-studied allosteric enzyme. The substrates of the ATCase reaction are carbamoyl phosphate (CbmP) and aspartate; the products are N-carbamoylaspartate plus orthophosphate. The following step in the pathway is cyclization of N-carbamoylaspartate to dihydroorotate. Gerhart and Pardee found in 1962 that the enzyme displays sigmoidal kinetics, meaning the reaction velocity vs. substrate concentration curve has a sigmoidal form. The enzyme shows a strong preference for binding of CbmP first. Once CbmP is bound, the affinity of the enzyme for aspartate is increased markedly, and aspartate shows a positive cooperative effect on catalysis.

The reaction likely proceeds through a tetrahedral transition state after the α-amino group of aspartate attacks the carbonyl carbon of carbamoyl phosphate, and prior to the expulsion of the orthophosphate leaving group. Note that CbmP is a fairly high-energy phosphoric anhydride, making this reaction energetically favorable (energy is required to synthesize CbmP).

The first structure of ATCase was determined by X-ray crystallography in 1982 by William Lipscomb and associates (2.6 A resolution). It showed a dodecameric (12 subunit) quaternary structure constructed of two catalytic trimers separated along a vertical axis corresponding to a three-fold symmetry axis, with the catalytic trimers bridged by three regulatory dimers. This dodecameric structure, referred to as the holoenzyme (to distingish it from catalytic trimers and regulatory dimers), was fully consistent with biochemical studies of ATCase. Catalytic trimers and regulatory dimers can be isolated from intact holoenzyme by treatment with mercurial reagents followed by separatory procedures. The catalytic trimers have full catalytic activity and show no cooperative behavior.

An extremely useful inhibitor of ATCase known as PALA is a bisubstrate analog. PALA is a non-hydrolyzable phosphonate compound which is similar to CbmP + aspartate, except for a missing amino group. PALA has a K_d of about 10 nM for ATCase, meaning it binds the enzyme quite strongly. The crystal structure of ATCase in complex with the bisubstrate analog PALA was used not only to infer the nature of enzyme-substrate contacts, but also (in comparison to the structure of the free enzyme) to in the effort to define the nature of the structural changes accompanying allosteric effects. The active site is formed at the interface between subunits within a catalytic trimer. If the structures of unliganded ATCase holoenzyme and PALA-bound ATCase holoenzyme represent the T and the R states, respectively, then the change from T state to R state is associated with an increase in separation of the catalytic trimers by 12 Å, as well as a relative rotation of 10º. This dramatically reduces the interactions between c chains in different opposing trimer. The regulatory chains also rotate, accommodating the larger distance between the catalytic trimers. The individual c chains within the catalytic trimer also undergo a structural transition. The two domains within the c subunit approach one another more closely by a hinge motion of about 8º, resulting in a closure at the active site of about 2 Å. The closure of the active site is stabilized by interdomain bridging interactions.

Furthermore, ATCase shows heterotropic effects. Cytidine triphosphate (CTP), which is a product of this pathway, has a negative allosteric effect, while adenosine triphosphate, ATP, has a positive allosteric effect. These effects are analogous to the Bohr effect in hemoglobin. CTP and ATP compete for the same regulatory binding site. The negative allosteric effect of CTP is an example of feedback inhibition, a typical mechanism by which biosynthetic pathways are regulated.

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.

Finally, we'll examine the regulation of a kinase known as protein kinase A. In this case, regulation is via a noncovalent mechanism: the binding of the small molecule ligand 3',5'-cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase, and the binding of cAMP to regulatory subunits of PKA activates the enzyme by triggering the dissociation of the regulatory subunits from the catalytic subunits

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.

Activation of enzymes by specific proteolytic cleavage

Proteolytic activation of pancreatic serine proteases.

We finish our consideration of regulatory strategies by learning about a case of activation by proteolytic cleavage that is of obvious physiological importance - the blood clotting cascade. A series of proteolytic activation events, each of which is similar to the activation of the zymogen forms of the pancreatic serine proteases, leads to the formation of blood clots in response to injury or trauma. This blood clotting pathway has become the classic example of a zymogen activation cascade. A key feature of this cascade is the amplification of an initial triggering event into a major physiologic response.

Intrinsic pathway

* Triggered by the "non-physiological surface" of an injury
* Begins with activation of Factor XII (Hageman factor)

Extrinsic pathway

* Trauma triggers activation of Factor VII
* Release of lipoprotein called tissue factor follows

Final common pathway

* Intrinsic and extrinsic pathways converge, both activating Factor X
* The end result is the conversion of fibrinogen to fibrin by thrombin

Thrombin - yet another serine protease

In the blood clotting cascade, the active form of the serine protease thrombin is responsible for the conversion of fibrinogen to fibrin. A clot forms when fibrin monomers formed by the action of thrombin assemble into ordered fibrous arrays. Thrombin shows a specificity for cleavage of the peptide bond between Arg and Gly, suggesting a similarity to trypsin. Indeed, the B chain of thrombin shows sequence similarity to trypsin, and the X-ray structure of thrombin reveals all the hallmarks of a serine protease: the catalytic triad, oxyanion hole, and specificity pocket. The latter, like trypsin, has an aspartate residue at its bottom that interacts favorably with the Arg at P₁ of the substrate.

Thrombin arises from the processing of an inactive precursor, or zymogen, called prothrombin. Prothrombin is a 582-residue polypeptide whose first 274 residues constitute the large "pro" region. Processing (proteolytic cleavage) by Factor Xa (stimulated by Factor Va) corresponds to cleavage of the Arg274-Thr275 and Arg323-Ile324 peptide bonds. The pro region is released, while the two fragments of the mature polypeptide remain associated and are covalently joined by a disulfide bond.

The pro region of thrombin contains (near the N-terminus) a number of modified Glu residues that contain an extra carboxyl group. These residues, called γ-carboxyglutamate (three-letter abbreviation Gla), act as effective chelators of Ca²⁺ ions. This property of prothrombin is essential for the proper functioning of the clotting cascade. This is because the binding of calcium ions by prothrombin anchors it to phospholipid membranes derived from platelets following injury. This properly localizes prothrombin in proximity to its activating factors.

Learning objectives

• List and provide examples of each of the four regulatory strategies discussed.
• Apply the concerted (MWC) model to explain homotropic and heterotropic effects in allosteric enzymes.
• Explain the metabolic logic of heterotropic effects of CTP and ATP on ATCase activity.
• Propose a reasonable mechanism for the reaction catalyzed by ATCase.
• Define kinase and protein kinase. Name the common residue targets of protein kinases.
• Describe the how phosphorylation of protein targets alters their biochemical properties..

Page updated 10-30-2010

References

1. Berg, Tymoczko, and Stryer. Biochemistry (BTS): 6th edition (2007, Freeman) pp.275-282
2. Schachman HK. (2000). Still looking for the ivory tower. Annu Rev Biochem 69: 1-29.