Presentation on theme: "COVALENT ENZYME REGULATION Both reversible and irreversible covalent modification of enzymes play important roles in regulation of enzyme function. This."— Presentation transcript:
COVALENT ENZYME REGULATION Both reversible and irreversible covalent modification of enzymes play important roles in regulation of enzyme function. This lecture will cover: 1. Reversible covalent modification. The modulation of enzyme activity by the attachment or release of small groups plays a very important role in metabolic control. Probably the most universal, and certainly the most well understood, is the phosphorylation of specific serine, threonine or tyrosine groups. We will discuss protein phosphorylation, regulation and its effects on enzyme structure and function 2). Irreversible covalent modification. Proteolytic cleavage of specific peptide bonds is often used to activate enzymes. Since proteolysis is essentially irreversible, turning the activity off requires another mechanism, often binding of inhibitory proteins. Examples of enzymes activated by proteolytic cleavage and the role of enzyme cascades will be discussed. Reading: Lippincott, ch. 5, section VIII
1) Reversible covalent modifications. Reversible covalent modifications require expenditure of energy and are often used in signaling from extracellular messages. In contrast, noncovalent interactions are reversible with no metabolic energy expended and sense conditions within a cell. Reversible covalent modifications that are known to alter enzyme activity include: a) Phosphorylation of serine, threonine or tyrosine and less frequently aspartate and histidine residues. b) Acetylation of lysine or amino terminal groups. c) Methylation of glutamate or aspartate residues d) Nucleotidylation of tyrosine residues e) ADP ribosylation primarily of arginine residues. Most well understood of these reactions, and probably the most ubiquitous in eukaryotic cells, is the phosphorylation reaction, which is based on the simple addition and removal of inorganic phosphate.
Lysine Acetylation Acetylation of lysines in histones is important in regulation of gene expression. Addition of the acetyl group to a lysine removes its positive charge, weakening the binding of histones to the negatively charged DNA which, apparently, results in a conformation more favorable for transcription NH 3 + N H C CH 3 O C O S CoA HS CoA Lysine Acetylated Lysine Acetyl Coenzyme A Coenzyme A CC CC
The energy for reversible phosphorylation is derived from ATP hydrolysis. Protein Phosphorylation: Enzymes catalyzing the transfer of a phosphate from ATP to a protein are known as kinases and those catalyzing the hydrolytic removal of the phosphate group are known as phosphatases.
Kinases and Phosphatases come in two major classes - those that act specifically on serine and threonine residues and those that act on tyrosine residues. HCH CC CH 3 HC CC HCH CC OH SerineThreonine Tyrosine OPO 3 2 -
Regulation of Protein Kinases Regulation by phosphorylation requires that the kinases and phophatases must, in turn, be regulated. Regulation of kinases, and probably phosphatases as well, most often involves one or more of three regulatory strategies: a) interaction with peptides or subunits whose binding may depend upon chemical messengers such as calcium or cyclic AMP. b) phosphorylation itself is a very common mechanism for regulation of protein kinases (an enzyme that catalyzes this reaction would be known as a kinase kinase). c) localization to particular cellular components. We will provide examples of the first two strategies shortly, using the binding of cyclic AMP to PKA regulatory subunits as an example of the first strategy and the effects of phosphorylation on the insulin receptor tyrosine kinase as an example of the second strategy. An additional important strategy that has recently become more appreciated is the targeting of kinases to specific cellular locations. This can be used to limit the effects of kinases with broad specificity only to desired locations. A number of proteins and protein domains have been implicated in such targeting.
Phosphorylation in the regulation of glycogen metabolism The role of reversible covalent modification was first shown to be important in the control of glycogen metabolism. Glycogen is used by the body as a readily mobilized storage of glucose. Glycogen synthase catalyses synthesis of glycogen while Glycogen phosphorylase catalyses the stepwise removal of glucose units from glycogen. Synthesis and degradation of glycogen is coordinated. Shown to the right are schematic pathways resulting from epinephrine binding to its receptor on the plasma membrane. The first three steps are identical, with both leading to protein phosphorylation. However, phosphorylation of phosphorylase leads to its activation, whereas phosphorylation of glycogen synthase leads to its inactivation.
Enzyme Cascades As is evident from the previous slide, the same protein kinase that phosphorylates and activates phosphorylase kinase also phosphorylates glycogen synthase but in this case causes inactivation. Generally enzymes that are involved in degradation are activated by phosphorylation and those involved in synthesis are inhibited by phosphorylation. The activation by an enzyme cascade considerably amplifies the hormone signal. Three steps are used to activate glycogen breakdown and two deactivate glycogen synthesis. If glycogen synthase and phosphorylase were directly controlled by binding epinephrine, more than one thousand times as much hormone would be needed to elicit the same response obtained through the cascades.
cAMP dependent kinase (PKA): The kinase that mediates the hormone signal is activated by cAMP and is known as cAMP dependent kinase (cAPK) or protein kinase A (PKA). Although first discovered in the glycogen metabolism pathway, PKA is involved in a large number of activities as the major mediator of cAMP action. PKA specifically phosphorylates serine or threonine residues preferably in the following amino acid sequence: Arg - Arg - X - Ser (or Thr) - Y, ( where X is a small residue and Y is a large hydrophobic residue). Most kinases rely primarily on the amino acid sequence surrounding the phosphorylation site for their specificity. Activation of PKA occurs when cAMP binds to the regulatory subunits. In the absence of cAMP, the regulatory subunits bind tightly to the catalytic subunits forming a heterotetramer. A portion of the regulatory subunit with the sequence: Arg - Arg - Gly - Ala - Ile binds in the active site of the catalytic subunit. This sequence matches the preferred sequence except for the actual phosphorylation site, and thus binds tightly to the active site without being modified. The binding of cAMP to the regulatory subunits allosterically causes dissociation, which allows the catalytic subunits to attain an active conformation by freeing the active site. Cyclic AMP (cAMP)
The crystal structure of the catalytic subunits of PKA was the first determined for a protein kinase. The structure has two lobes separated by a deep cleft that contains the active site (see right). The 240 residue catalytic core shares sequence homology with hundreds of other kinases suggesting that all these kinases will have a similar structure. Indeed, crystal structures for many serine/threonine kinases and tyrosine kinases demonstrate a common three- dimensional structure for the catalytic core of protein kinases. The structure of PKA was determined bound with a peptide inhibitor revealing structural determinants of sequence specificity. This peptide has an amphipathic helix that contributes to stability of the inhibitor-kinase complex and, more importantly, contains the sequence Arg-Arg-Asn- Ala-Ile that binds to the active site. The structure shows that both Arg residues are involved in ionic interactions with glutamate residues and the Ile residue packs in a hydrophobic groove formed by two Leu residues in the kinase. Thus the specificity of this kinase for these three residues can be readily understood. Structure of PKA catalytic subunit, with bound peptide inhibitor (dark red) Interactions of bound inhibitor (stick bonds) with PKA catalytic core residues. Arg Asn Ala Ile Glu 127 Glu 170 Glu 230 Leu 205 Leu 198
Structural and functional effects of protein phosphorylation Phosphorylation results in the addition of a doubly negatively charged group (at neutral pH) to a previously uncharged amino acid. Although this is a small group, it can profoundly impact protein function. Three different ways this occurs include: a) direct interference with the active site, b) conformational change in the enzyme or c) creation of binding sites. Examples of these mechanisms are given below. a) Direct steric interference is observed in the structures of isocitrate dehydrogenase (IDH) which show that phosphorylation of Ser 113 in the active site directly interferes with binding of the isocitrate substrate (see below). Active site of IDH showing binding of isocitrate substrate isocitrate Ser 113 Phosphate Active site of IDH showing phosphorylated Ser 114
b) Enzymatic conformational change upon phosphorylation is dramatically evident from the structures of the insulin receptor kinase (IRK) determined in the phosphorylated and unphosphorylated states as shown below. (Figures courtesy of Dr. Stevan Hubbard, NYU.) Unphosphorylated IRK (left) and phosphorylated IRK (right). The surface of IRK is shown in gray, except for the N-terminal domain in white in the left figure. The activation loop in unphosphorylated IRK blocks binding of a substrate polypeptide chain, but moves out of the way, to the right, upon phosphorylation at three tyrosines. Thus, triple phosphorylation of this loop results in activation of kinase activity.
c) Creation of binding sites – SH2 domains. Peptides containing a phosphorylated tyrosine residue are bound by an important signaling protein module known as an SH2 (src homology) domain. These domains are about 100 residues in length that fold into a central anti-parallel - sheet surrounded by two -helices. SH2 domains are found in many diverse protein molecules and function to bind phosphorylated tyrosines in a specific polypeptide sequence. The structure of an SH2 domain (light gray) with a bound peptide (green) is shown to the right. The phosphorylated tyrosine is shown in red, with the phosphate group in green. Key residues forming a binding pocket are shown, including an absolutely conserved arginine (R32) whose interaction with the phosphate group is critical for recognizing phosphorylated tyrosine residues. R32
In general, a protein kinase is an enzyme that: A) Catalyzes the transfer of a phosphate group from cAMP to a protein side chain B) Catalyzes the transfer of a phosphate group from ADP to a protein side chain C) Catalyzes the transfer of a phosphate group from ATP to a protein side chain D) Catalyzes the cleavage of a specific peptide bond E) Catalyzes the removal of a phosphate group from a protein Which of the following is NOT an important mechanism by which phosphorylation alters protein function? A) Phosphorylation can cause direct steric interference by the phosphate at the active site B) Phosphorylation at serine residues will induce proteolytic cleavage of the peptide bond amino terminal to the serine C) Phosphorylation can induce protein conformational changes D) Phosphorylation at tyrosine residues can create binding sites that are recognized by SH2 domains E) Phosphorylation results in the addition of a doubly negatively charged group that can alter the local protein structure
II) Irreversible covalent modification (limited proteolysis) In a number of cases, it is necessary to synthesize an enzyme in an inactive state and activate it later by selective cleavage of one or more peptide bonds. The inactive precursors are termed zymogens or proenzymes. Proteolytic cleavage generally occurs at surface loops, rather than secondary structural elements. Generally, the proteolytic site is amino terminal relative to the active site of the protein. Since the polypeptide is synthesized in the amino to carboxy direction, the protein can, thus, avoid gaining catalytic activity as it folds during synthesis. A) Digestive enzymes The digestive enzymes are classic examples for which activation of enzyme activity occurs by selective cleavage. Synthesis as inactive zymogens permits export to the digestive tract before the destructive catalytic powers of these enzymes are unleashed on the synthesizing cells. Site of synthesisZymogenActive Enzyme StomachPepsinogenPepsin PancreasChymotrypsinogenChymotrypsin* PancreasTrypsinogenTrypsin* PancreasProcarboxypeptidaseCarboxypeptidase PancreasProelastaseElastase* Gastric and pancreatic digestive enzymes *Serine Protease
Chymotrypsin, trypsin and elastase are examples of the major class of proteases known as serine proteases (discussed in lecture 15) which are so named because they have a highly reactive serine at the active site that is essential for catalytic activity. Different members of the serine protease family have very similar catalytic sites, but distinct specificity due to flanking regions that bind various amino acid side chains with differing affinity. The first step in the activation of pancreatic zymogens is a very specific cleavage of a small amount trypsinogen by enteropeptidase. This occurs after trypsinogen secretion from the pancreas into the duodenum. These newly formed trypsin molecules activate other trypsinogen molecules and other zymogens.
Activation of enzymatic activity does not require major changes in the protein structure. Chymotrypsinogen is activated by cleavage of the peptide bond between residues 15 and 16, with the N-terminal peptide remaining attached to the rest of the protein due to a disulfide linkage. (Additional cleavages can take place but are not essential for catalytic activity.) Cleavage does not lead to large molecular changes, but rather to subtle changes that alter the active site geometry so as to maximally stabilize the transition state.
A much larger protein, the 53 kDa 1-antitrypsin, plays a similar role in the inactivation of elastase. Elastase is secreted by neutrophils as part of the inflammatory process. In order to restrict its activity to the site of infection, it is turned off by 1-antitrypsin. The importance of its role is apparent from misfolding mutants. One mutation (glu 53 -> lys) results in reduced secretion from the liver, lowering the serum level of 1-antitrypsin to 15% of that in normal individuals. (The lowered secretion stems from the pathological polymerization of the mutant, causing cirrhosis of the liver, as discussed in lecture 5.) The resulting excess elastase activity in the serum destroys the alveolar walls in the lungs and leads to emphysema. Cigarette smoking increases the propensity of heterozygotes for the above mutation to develop emphysema, as a result of oxidation of methionine 358 of 1-antitrypsin (see right). The addition of a single oxygen atom in this residue drastically decreases the affinity of the inhibitor for elastase. Since activation by selective peptide bond cleavage is irreversible, alternate mechanisms must exist for turning off enzyme activity. Specific protease inhibitors are available for this function. Pancreatic trypsin inhibitor (PTI) is a small (6-kDa) protein that binds to trypsin tightly in its active site. Binding is very tight due to precise structural complementarity between PTI and the active site of trypsin. Inactivation of digestive enzymes -CH 2 - CH 2 – S – CH 3 Methionine -CH 2 - CH 2 – S – CH 3 O Methionine sulfoxide Oxidation
B) Blood Clotting. Blood clotting results from the participation of nearly 20 different substances involved in two different cascades of proteolytic reactions. The use of cascades serve to amplify small stimuli to major physiological responses. The two cascades are known as the intrinsic and extrinsic pathways. The intrinsic pathway covers five proteolytic reactions and is mediated by components found in the plasma. The extrinsic pathway comprises four proteolytic steps and includes factors found in tissues. Both pathways meet at the conversion of prothrombin to thrombin which then converts fibrinogen to fibrin. The active clotting factors are, except for fibrin and factor XIIIa, serine proteases similar to the members of the trypsin family. The final purpose of these cascades is the conversion of fibrinogen to fibrin to form a clot.
Formation of Fibrin Clot: The ultimate purpose of the blood clotting pathways is to trigger the conversion of fibrinogen into fibrin which then polymerizes to form a clot. Fibrinogen is very abundant, comprising 2-3% of plasma protein, and is formed from three pairs of polypeptide chains into a highly disulfide linked elongated complex (~450A in length). Crystal structure of bovine fibrinogen. The A chains are shown in green, B chains in blue and in red. Schematic diagram of fibrinogen. At the molecular center are four “fibrinopeptides” (two A and two B) at the amino end of the and chains whose abundance of negative charge inhibits polymerization. Conversion to fibrin results from thrombin cleavage at specific Arg-Gly peptide bonds, which releases these peptides and results in the center region becoming more positively charged. Thrombin cleavage Fibrin Clot. Following removal of the negatively charged A and B fibrinopeptides, charge interactions between fibrin molecules stabilize the formation of the arrangement illustrated below. This half-staggered arrangement, with a fiber repeat of 225Å, is the fundamental assemblage of the fibrin clot.
The fibrin clots are considered "soft clots" and can be turned into more stable "hard clots" cross-linking lysine and glutamine residues in a transamidation reaction by factor XIIIa. (Factor XIIIa is also known as fibrin-stabilizing factor (FSF) or transglutaminase.) Thrombin catalyzes the cleavage of the Arg-Gly bond in fibrinogen that initiates clot formation. Its structure shows very striking similarity with those of the pancreatic serine proteases especially in the active site. The major difference is its extreme specificity in cleaving only certain Arg - Gly bonds. Lys-CH 2 - CH 2 -CH 2 - CH 2 -NH 2 + NH 2 -C -CH 2 - CH 2 - Gln Lys-CH 2 - CH 2 -CH 2 - CH 2 -NH -C -CH 2 - CH 2 - Gln Activated FSF NH 3 O O
Because of the amplification effects of the cascade reactions, the early factors are only present at very small concentrations ( g/ml) compared with the 3mg/ml for fibrinogen. This, along with their lack of stability, has complicated analysis of these proteins. Much of the early knowledge of the clotting reaction came from bleeding disorders. The most well known of these is classic hemophilia, a sex-linked disorder in which Factor VIII is missing or has a very reduced activity. This factor stimulates the activation of Factor X by Factor IX. Treatment of hemophiliacs used to require that large amounts of blood be fractionated to obtain reasonable quantities of coagulation factors. This was expensive and risky. The availability of recombinant clotting factors now minimizes these risks. Several serine proteases are involved in the activation of plasminogen including urokinase and tissue-type plasminogen activator (TPA). Plasminogen activators are of considerable medical significance and are used to promote rapid dissolving of blood clots responsible for heart attacks and strokes. Both recombinant TPA and streptokinase have been successfully used for this purpose. Blood clots are only meant to be temporary patches. Once structural integrity is returned to the damaged area, they must be dissolved. The protein responsible for lysis of the clot is, once again, a serine protease, this time one named plasmin. The open mesh-like structure of a blood clot allows ready access to plasmin, which is formed by the proteolytic cleavage of plasminogen.
The pancreatic digestive enzymes are activated by: A) Cleavage of specific peptide bonds by trypsin B) Binding of BPG to basic residues in the subunit interface C) Cleavage of C-terminal residues by carboxypeptidase D) Phosphorylation of serine and threonine residues E) Methylation of aspartate residues TPA is of medical importance because it: A) Directly dissolves fibrin based blood clots. B) Cleaves plasminogen to form plasmin which then dissolves blood clots. C) Phosphorylates and activates plasminogen which then dissolves blood clots. D) Prevents massive bleeding by stimulating blood clotting. E) Assists plasmin in dissolving blood clots by opening up cleavage sites.
Summary Points on Covalent Enzyme Regulation: 1) Know the reactions catalyzed by protein kinases and phosphatases. 2) Understand the amplification effect of enzyme cascades. 3) Know the basis for activation of cAMP dependent Kinase (PKA). 4) Know that there are three important basic mechanisms by which phosphorylation alters protein function including: steric interference, conformational change as a result of introduction of a doubly negatively charged group and creation of binding sites. 5) Understand that activation of digestive enzymes and blood clotting factors by proteolysis may be mediated by localized subtle structural changes that have large functional consequences. 6) Know that enzymes activated by proteolysis are usually inactivated by binding inhibitors. 7) Know what is meant by a serine protease. 8) Know that activation of fibrinogen to form fibrin clots occurs as a result of proteolysis of specific Arg-Gly bonds by thrombin. 9) Know that TPA is a serine protease that promotes lysis of blood clots by proteolytic cleavage of plasminogen to form plasmin