CHAPTER 10: REGULATORY STRATEGIES. Traffic signals control the flow of traffic

Transcription

1 CHAPTER 10: REGULATORY STRATEGIES Traffic signals control the flow of traffic

2 INTRODUCTION CHAPTER 10 The activity of enzymes must often be regulated so that they function at the proper time and place. Enzymatic activity is regulated in five principal ways: 1. Allosteric Control Enzyme activity is controlled by the binding of small signal molecules at regulatory sites Allosteric proteins show the property of cooperativity: activity at one functional site affects the activity at others Aspartate transcarbamoylase (ATCase) Hemoglobin

3 INTRODUCTION CHAPTER Multiple Forms of Enzymes Isozymes are homologous enzymes within a single organism - Catalyze the same reaction - Slightly different in structure and catalytic/regulatory properties - Expressed in a distinct place or at a distinct stage of development 3. Reversible Covalent Modification Alters the catalytic properties of many enzymes - Phosphorylation by protein kinases - Dephosphorylation by protein phosphatases - Protein kinase A

4 INTRODUCTION CHAPTER Proteolytic Activation Activation of proenzymes (zymogens) by proteolytic cleavage - Chymotrypsin, trypsin, and pepsin are activated by this mechanism - Blood clotting is due to a cascade of zymogen activations 5. Controlling the Amount of Enzyme Present Enzyme activity is regulated by adjusting the amount of enzyme present - This regulation usually takes place at the level of transcription

5 10.1 ASPARTATE TRANSCARBAMOYLASE CHAPTER 10 The enzyme catalyzes the 1 st step in the biosynthesis of pyrimidines: The condensation of aspartate and carbamoyl phosphate The committed step in the pathway for pyrimidine nucleotide such as CTP Fig 10.1 ATCase reaction.

6 10.1 ASPARTATE TRANSCARBAMOYLASE CHAPTER 10 It was found that ATCase is inhibited by CTP Feedback inhibition The inhibition of an enzyme by the end product of the pathway Allosteric regulation CTP is structurally quite different from the substrates CTP must bind to a site distinct from the active site Fig 10.1 CTP inhibits ATCase.

7 SIGMOIDAL KINETICS The dependence of the reaction rate on [Asp] Sigmoidal curve 10.1 ASPARTATE TRANSCARBAMOYLASE Cooperativity the binding of substrate to one active site increases the activity at the other active sites Does not follow Michealis-Menten kinetics The majority of allosteric enzymes display sigmoidal kinetics Cooperation between subunits in hemoglobin Fig 10.3 ATCase displays sigmoidal kinetics.

8 10.1 ASPARTATE TRANSCARBAMOYLASE CATALYTIC AND REGULATORY SUBUNITS ATCase can be separated into regulatory (r) and catalytic (c) subunits by treatment with p-hydroxymercuribenzoate (p-hmb) Catalytic subunit (c 3 ), three chains (34 kd each) Regulatory subunit (r 2 ), two chains (17 kd each) Native p-hmb treated Fig 10.4 Modification of cysteine residues. Fig 10.5 Ultracentrifugation studies of ATCase.

9 10.1 ASPARTATE TRANSCARBAMOYLASE CATALYTIC AND REGULATORY SUBUNITS The larger subunit displays catalytic acitivity; unresponsive to CTP, not sigmoidal kinetics catalytic subunit The smaller subunit can bind CTP; no catalytic acitivty regulatory subunit The subunits combine rapidly when they are mixed 2 c r 2 c 6 r 6 The reconstituted enzyme has the same structure and allosteric/catalytic properties as those of the native enzyme

10 STRUCTURE OF ATCASE Two catalytic trimers are stacked one on top of the other Significant contacts between the catalytic and the regulatory subunits 10.1 ASPARTATE TRANSCARBAMOYLASE 4-Cys bound Fig 10.6 Structure of ATCase.

11 10.1 ASPARTATE TRANSCARBAMOYLASE STRUCTURE OF ATCASE A bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA) was used for the ATCase-substrate analog complex structure PALA is a potent inhibitor for ATCase Fig 10.7 PALA, a bisubstrate analog.

12 10.1 ASPARTATE TRANSCARBAMOYLASE STRUCTURE OF ATCASE The structure of the ATCase-PALA complex PALA binds at site lying at the boundaries between pairs of c chains within a catalytic trimer Fig 10.8 The active site of ATCase.

13 10.1 ASPARTATE TRANSCARBAMOYLASE STRUCTURE OF ATCASE Remarkable change in quaternary structure on PALA binding ATCase shows two distinct quaternary forms: the T (tense) state and the R (relaxed) state Fig 10.9 The T-to-R state transition in ATCase.

14 10.1 ASPARTATE TRANSCARBAMOYLASE STRUCTURE OF ATCASE How can we explain the enzyme s sigmoidal kinetics? In the absence of substrate, almost all the enzyme molecules are in the T state Low affinity for substrate; low catalytic activity The substrate binding to one active site Increases the likelihood that the entire enzyme shifts to the R state with higher affinity Conversion of the enzyme into the R state causes more substrate to bind to the active site Cooperativity

15 10.1 ASPARTATE TRANSCARBAMOYLASE STRUCTURE OF ATCASE The sigmoidal curve can be pictured as a composite of two Michealis-Menten curves The T-to-R transition is taking place within a narrow range of substrate concentration Makes it possible to respond to small changes in substrate concentration Fig 10.8 Basis for the sigmoidal curve.

16 CTP EFFECTS ON THE T-TO-R EQUILIBRIUM CTP inhibits the action of ATCase Structure of CTP-bound ATCase The enzyme is in the T state CTP binds to the regulatory domain The binding site is more than 50 Å from the active site 10.1 ASPARTATE TRANSCARBAMOYLASE Fig CTP stabilizes the T state.

17 10.1 ASPARTATE TRANSCARBAMOYLASE CTP EFFECTS ON THE T-TO-R EQUILIBRIUM The binding of CTP shifts the equilibrium toward the T state Stabilizes the T state Decreases net enzyme activity Increases the initial phase of the sigmoidal curve Fig The R state and the T state are in equilibrium. Fig Effect of CTP on ATCase kinetics.

18 10.1 ASPARTATE TRANSCARBAMOYLASE CTP EFFECTS ON THE T-TO-R EQUILIBRIUM ATP increases the reaction rate at a given Asp concentration Competes with CTP for binding to regulatory sites High ATP means high purine: needs to make more pyrimidine for balance High ATP means high energy for mrna synthesis and DNA replication: leads to the synthesis of more pyrimidines Fig Effect of ATP on ATCase kinetics.

19 10.2 REGULATORY STRATEGIES IN ISOZYMES CHAPTER 10 Isozymes are enzymes that differ in AA sequence yet catalyze the same reaction Have different kinetic parameters or respond to different regulatory molecules Permits the fine-tuning of metabolism to meet the needs of a given tissue or developmental stage Lactate dehydrogenase Involved in glucose synthesis and metabolism Two isozymic peptides exist: H isozyme and M isozyme; 75% identical sequence Functions as a tetramer: many different combinations exist

20 10.2 REGULATORY STRATEGIES IN ISOZYMES CHAPTER 10 The isozymes (H 4 & M 4 ) are functionally different H 4 : inhibited by high levels of pyruvate; functions in aerobic environment M 4 : no inhibition by high levels of pyruvate; functions in anaerobic environment H 2 M 2 has intermediate properties M 4 H 4 The rat heart LDH isozyme profile The tissue-specific forms of lactate dehydrogenase Days before (-) & after (+) birth in adult rat tissues Fig Isozymes of lactate dehydrogenase.

21 10.3 COVALENT MODIFICATION CHAPTER 10

22 10.3 COVALENT MODIFICATION KINASES AND PHOSPHATASES Phosphorylation is a regulatory mechanism used in every metabolic process in eukaryotic cells 30% of eukaryotic proteins are phosphorylated Catalyzed by protein kinases Protein kinases are one of the largest protein families: more than 500 protein kinases in human beings Fine-tuned regulation according to a specific tissue, time, or substrate ATP is the most common donor Ser, Thr, and Tyr are the acceptors

23 KINASES AND PHOSPHATASES 10.3 COVALENT MODIFICATION

24 KINASES AND PHOSPHATASES Tyrosine kinases Play pivotal roles in growth regulation Mutations are observed in cancer cells Ser/Thr kinases 10.3 COVALENT MODIFICATION

25 10.3 COVALENT MODIFICATION KINASES AND PHOSPHATASES Substrate specificity of Ser/Thr kinases Some kinases phosphorylate a single protein or several closely related ones Multifuncational kinases modify many different targets - Recognize the consensus sequence, Arg-Arg-X-Ser/Thr-Z; X a small residue; Z, a large hydrophobic residue

26 KINASES AND PHOSPHATASES 10.3 COVALENT MODIFICATION Protein phosphatases remove phosphoryl groups attached to proteins Turn off the signaling pathways activated by kinases One class of conserved phosphatases (PP2A) suppresses the cancerpromoting activity of certain kinases Irreversible / unidirectional Take place only by enzymes

27 10.3 COVALENT MODIFICATION PHOSPHORYLATION IS HIGHLY EFFECTIVE Phosphorylation is a highly effective means of regulating the activities of target proteins for several reasons: 1. The free energy of phosphorylation is large 20 ~ 30 kj/mol (about 5 kcal/mol) A free energy change of 5.69 kj/mol (1.36 kcal/mol) corresponds to a factor of 10 in an equilibrium constant Phosphorylation shifts the equilibrium by Adds two negative charges to a modified protein These charge can cause a large conformational change Such structural changes can markedly alter substrate binding and catalytic activity

28 10.3 COVALENT MODIFICATION PHOSPHORYLATION IS HIGHLY EFFECTIVE Phosphorylation is a highly effective means of regulating the activities of target proteins for several reasons: 3. A phosphoryl group can form three or more H-bonds Can make specific interaction 4. Phosphorylation and dephosphorylation can take place in less than a second or over a span of hours The kinetics can be adjusted to meet the timing needs of a physiological process

29 10.3 COVALENT MODIFICATION PHOSPHORYLATION IS HIGHLY EFFECTIVE Phosphorylation is a highly effective means of regulating the activities of target proteins for several reasons: 5. The effects of phosphorylation can be highly amplified A single activated kinase can phosphorylate hundreds of target proteins in a short interval 6. ATP is a cellular energy currency The use of ATP as a phosphoryl-group donor links the energy status of the cell to the regulation of metabolism

30 CYCLIC AMP ACTIVATES PROTEIN KINASE A Cyclic AMP (camp) is an intracellular messenger formed by the cyclization of ATP camp (>10 nm) activates a key enzyme, protein kinase A (PKA) The activated PKA alters the activities of target proteins by phosphorylation Most effects of camp in eukaryotic cells are achieved through the activation of PKA 10.3 COVALENT MODIFICATION

31 10.3 COVALENT MODIFICATION CYCLIC AMP ACTIVATES PROTEIN KINASE A PKA consists of two kinds of subunits: A 49-kD regulatory (R) subunit and a 38-kD catalytic (C) subunit In the absence of camp, PKA exists in a R 2 C 2 form The binding of camp to the R subunit relieve its inhibition of the C subunit Each R chain contains the sequence Arg-Arg-Gly-Ala-Ile (pseudosubstrate sequence), which occupies the catalytic site of C The binding of camp allosterically removes the sequence resulting in the activation of C Fig Regulation of PKA.

32 THE CRYSTAL STRUCTURE OF PKA 10.3 COVALENT MODIFICATION The X-ray crystal structure of PKA complexed with ATP and a 20-residue peptide inhibitor Two lobes: the smaller lobe, contacts with ATP-Mg 2+ ; the larger lobe, binds to the peptide substrate Residues 40 to 280 are conserved in all known protein kinases Fig PKA bound to an inhibitor.

33 THE CRYSTAL STRUCTURE OF PKA 10.3 COVALENT MODIFICATION The bound peptide in the structure occupies the active site Fig Binding of pseudosubstrate to protein kinase A. Two guanidinium groups interact with three carboxylates in the active site Two Leu make a hydrophobic site to accommodate the Ile of the peptide The binding of camp allosterically blocks this interaction resulting in the activation of C

34 10.3 COVALENT MODIFICATION GLEEVEC A tyrosine-kinase inhibitor used in the treatment of multiple cancers, most notably Philadelphia chromosomepositive (Ph+) chronic myelogenous leukemia (CML) Received FDA approval in May 2001 Imatinib was one of the first cancer therapies; a paradigm for research in cancer therapeutics Imatinib has been cited as the first of the exceptionally expensive cancer drugs Time magazine cover of 28 May 2001 detailing Glivec as a 'cure' for cancer.

35 10.4 REGULATION BY PROTEOLYTIC CLEAVAGE Specific proteolysis is a common means of activating enzymes The inactive precursor is called a zymogen or a proenzyme Examples of regulation by proteolytic cleavage 1. Digestive enzymes CHAPTER 10

36 CHAPTER REGULATION BY PROTEOLYTIC CLEAVAGE Examples of regulation by proteolytic cleavage 2. Blood clotting thrombin, cascade 3. Some protein hormones Insulin is derived from proinsulin by proteolytic cleavage 4. Developmental processes: conversion of procollagenase into collagenase The metamorphosis of a tadpole into a frog: resorption of large amounts of collagen from the tail Break down of collagen in a mammalian uterus after delivery The conversion is precisely timed in these remodeling processes

37 CHAPTER REGULATION BY PROTEOLYTIC CLEAVAGE Examples of regulation by proteolytic cleavage 5. Apoptosis (programmed cell death) Eliminates damaged or infected cells and controls the shapes of body parts in the course of development Apoptosis is mediated by caspases, proteolytic enzymes Caspases are generated from procaspases by proteolytic cleavage Caspases function to cause cell death in most organisms ranging from C. elegans to human beings

38 10.4 REGULATION BY PROTEOLYTIC CLEAVAGE CLEAVAGE OF CHYMOTRYPSINOGEN Chymotrypsinogen Consisting of 245 AAs Synthesized in the pancreas Inactive form of chymotrypsin Cleaved by trypsin and converted into two peptides Self-cleaved to produce three peptide chains and two dipeptides Fig Proteolytic activation of chymotrypsin.

39 HOW THE CLEAVAGE ACTIVATES THE ZYMOGEN? The cleavage generates a new interaction between the N-terminal of Ile16 and the side chain of Asp194 The new interaction triggers a number of conformational changes Residues 187, 192, and 193 The changes generates the substrate-specificity site for hydrophobic groups Generation of the oxyanion hole Highly localized conformational change 10.4 REGULATION BY PROTEOLYTIC CLEAVAGE Fig Conformations of chymotrypsinogen (red) and chymotrypsin (blue).

40 10.4 REGULATION BY PROTEOLYTIC CLEAVAGE TRYPSIN IS THE COMMON ACTIVATOR Trypsin is the common activator of all the pancreatic zymogens Trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidase, and prolipase The formation of trypsin by enteropeptidase is the master activation step. Fig Zymogen activation by proteolytic cleavage.

41 10.4 REGULATION BY PROTEOLYTIC CLEAVAGE TRYPSIN IS THE COMMON ACTIVATOR The activity of trypsin is controlled by a pancreatic trypsin inhibitor The inhibitor is a 6-kd protein The dissociation constant of the complex is 0.1 pm Substrate analog Preorganized structure Cleavage rate of the inhibitor by trypsin is very slow (several months of half-life) Fig Interaction of trypsin with its inhibitor.

42 BLOOD CLOTTING 10.4 REGULATION BY PROTEOLYTIC CLEAVAGE Enzymatic cascades in biochemical systems Achieve a rapid response; an initial signal triggers a series of steps each of which is catalyzed by an enzyme Activation of 10 enzymes by an enzyme can activate 10 4 enzymes in 4 steps Blood clots are formed by a cascade of zymogen activations Small amounts of the initial factors suffice to trigger the cascade

43 10.4 REGULATION BY PROTEOLYTIC CLEAVAGE Begins with the activation of factor XII by contact with abnormal surfaces produced by injury BLOOD CLOTTING Fig Blood clotting cascade. Blood clotting is achieved by the interplay of the intrinsic, extrinsic, and final common pathways Triggered by trauma, which releases tissue factor (TF) TF forms a complex with VII, which initiates a cascade-activating thrombin Inactive form, in red Active form, in yellow Activated by thrombin, with *

44 HOMEWORK #1 FLUORESCENT PROTEINS History Structure Mechanism of the fluorescence Mechanism of the various colors Applications Due: the day of the 1 st exam (will be early April)

45 HOMEWORK #1 FLUORESCENT PROTEINS