Proteases: Hydrolysis of Peptide Bonds: Specificity and Mechanism

Transcription

1 Proteases: Hydrolysis of Peptide Bonds: Specificity and Mechanism C. Walsh Proteases (protein hydrolases) catalyze amide (peptide) bond hydrolysis in protein or peptide substrates: Cellular and physiologic functions: Hydrolysis of peptide bonds in the thousands of proteins inside and outside cells: control the dynamics of protein turnover Thermodynamics: Hydrolysis energetically favorable, K eq = Therefore proteolysis is irreversible (proteases are irreversible biological switches) and must be stringently controlled. Default position of regulated proteases must be in the off state: Diverse biological roles: Signal transduction: Polypeptide hormone, growth factor activation Proteolysis of I B to release NF B to go from cytoplasm to nucleus Defense: Blood coagulation and fibrinolysis (prothrombin and fibrinogen vs TPA, plasminogen) Cleavage of foreign proteins for MHC display Development: Proliferation : Fertilization; specification of dorsoventral polarity; Retroviral protease cleavage of polyproteins procollagen to collagen for self-assembly Proteolytic control of the cell cycle by cyclin Degradation Programmed Cell Death: homeostasis 1

2 Catalytic Features Specificity: continuum from one bond cleaved in a protein substrate, e.g in prohormone and procytokine activation, to digestive function on proteins. Proinsulin ProIL1 Insulin IL1 Rate Accelerations: up to 10 billion fold over nonenzymatic rates Mechanisms: four variants for activation of water molecules to attack and cleave peptide bonds in protein substrates: 2

3 Regulation and Control Temporal Control: produce proenzyme forms as inactive zymogen precursors, cleavable and activatable on demand (e.g trypsinogen, procarboxypeptidase; proacrosin; prococoonase) Spatial Control and Compartmentalization: 1. Protein inhibitors that bind tightly and produce inactive complexes on collision; find these antiprotease proteins both outside cells (antitrypsin, antithrombin 2 - macroglobulin at 10-4 M) and inside cells (IAPs) 2. Compartmentalization in organelles: lysosomal proteases for digestion of endocytosed proteins 3. Compartmentalization in subcellular machines: proteasomes, cylinders with protease active sites on the inside. Cascades of proteases: mechanisms for zymogen activation, amplification and explosive bursts of proteolysis, both extracellular (blood coagulation) and intracellular (caspases in programmed cell death) 3

4 Producer Cells make Zymogen Forms of Proteases Stomach Epithelia: Pepsinogen (aspartyl protease) Pancreatic acinar cells: Chymotrypsinogen (serine protease) Trypsinogen (serine protease) Proelastase (serine protease) Procarboxypeptidase (zinc protease) Zymogen activation: controlled proteolytic clipping of inactive precursor to active form of the protease: (single chain precursor to multi chain protease) Trypsin-mediated activation of chymotrypsinogen in the intestinal tract Autoproteolytic activation of HIV protease 4

5 Protease Classification (A) by site of action: exopeptidases (aminopeptidases, carboxypeptidases) vs endopeptidases (B) by reaction mechanisms and nature of active site residues involved in mechanism serine proteases cysteine proteases aspartyl proteases zinc (metallo) proteases The serine and cysteine proteases act directly as nucleophiles to attack the substrate, generatecovalent acyl enzyme intermediates. The aspartyl and zinc proteases activate water molecules as the direct attacking species on the peptide bond. Catalytic Mechanism of Chymotrypsin as a representative serine protease Key Issue: How to accelerate peptide bond hydrolysis by Catalytic Triad: His 57, Ser 95, Asp 102 typical inventory for serine proteases His 57 : acts in sequential steps, as Base, then Acid Catalyst for proton transfers Ser 195 : active site nucleophile, attacks peptide bond as ser alkoxide equivalent Asp 102 : orients His 57 side chain to H-bond to ser 195 5

6 Two Half Reactions: 1. Enzyme Acylation: Attack of ser 195 on bound substrate s peptide bond tetrahedral adduct acyl enzyme and release of amine product 2. Enzyme Deacylation: Attack of OH - on acyl enzyme tet adduct acid product 6

7 General Acid and General Base Catalysis (Proton Transfers) to Lower Activation Barriers and Accelerate Rates Accessibility of His and His-H + key for proton transfers in each step Same Fundamental Mechanism for Cysteine Proteases where Cysteine thiolate replaces serine alkoxide as active site nucleophile: Acyl enzyme intermediate is Peptidyl-S-Enzyme Specificity in Serine and Cysteine Proteases: Given the conseved catalytic triad inventory in all the serine and cysteine proteases and thus a common mechanism, how is specificity imposed? The Protease active sites have specificity pockets, subsites for binding the side chain of the amino acid on the acyl side of the peptide bond to be cleaved. Many proteases have even more extensive specificity subsites for recognizinf n+1,n+2 and/or n-1, n-2 residue side chains on protein substrates. 7

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9 For example while the identity and placement of the Asp-His-Ser catalytic triad in chymotrypsin, trypsin, and elastase is essentially identical, the specificity pockets differ, accommodating a hydrophobic aryl side chain (Cht), a cationic side chain (trypsin) or a small aliphatic side chain (elastase) The cysteine protease family (11 members) that act in apoptotic (programmed cell death) cascades all recognize the anionic side chain of an Asp residue and so cleave just downstream of such side chains, giving the name Caspase to this family; Cysteinyl proteases that cleave at asp residues. Aspartyl Proteases and Zinc Proteases Activate Water Molecules in the Active Site for Direct Attack on the Protein Substrate s Peptide Bond. Aspartyl Protease Catalytic Logic: Two aspartate residues in the enzyme active site, disposed on opposite faces of the peptide bond to be cleaved. One asp (Asp-COO - ) acts as general base to activate the attacking H-OH, the second asp (Asp-COOH) acts as general acid to protonate the departing amine product. The two asps act in complementary fashion for breakdown of the tetrahedral adduct. 9

10 The gastric protease pepsin has both asp residues in the same polype[tide chain while the 99 residue HIV protease has a single Asp. It becomes active as a dimer, providing the active site across the dimmer interface. This mechanism and architecture guided the development of HIV protease inhibitors that mimic the tetrahedral adduct. Zinc Protease Catalytic Logic: One atiom of Zn ++ coordinated specifically in active site to two his, one glu side chain brings superacid metal divalent cation into active site, with two additional coordination sites. In ground state, HOH bound and dissociates to Zn-OH, ready to attack the substrate peptide bond. Once the protein substrate is bound the active site zinc can coordinate to the carbonyl oxygen of the peptide bond to be attacked, lowering barriers electronically for OH attack. The tetrahedral adduct is coordinated to the active site zinc. A conserved glutamate side chain in the active site now acts as catalytic base to protonate the amine product as it leaves. Substrate analogs that have strong coordination sites for zinc (-SH, -COO) can be ppotent, selective inhibitors of zinc proteases. 10

11 Serine Proteases Size of the superfamily increases in eukaryotes: 1 gene in yeast, 18 genes in the worm, several hundred genes in Drosophila: Interest in proteomic approaches to identify function of the hundreds of serine proteases. Activity-based profiling approaches using covalent tethers attached to biotin for profiling (PNAS,96, 14694, 1999; Curr Opin Chem Biol 4, 663, 2000) Cysteine Proteases Family of cathepsins (11 members) in lysosomes; cathepsin K in bone remodeling, cathepsin L in antigen processing Family of 11 Caspases: Cysteine proteases that cleave at asp residues; cascade of caspase action in programmed cell death (initiator, amplifier, executioner proteases) Aspartyl Proteases Pepsin the first example HIV protease, a dimer with one Asp/subunit. Target for anti AIDS drugs: protease inhibitors such as saquinavir, ritonavir, indinavir - and -secretases in production of A 40 and A 42 in Alzheimers plaques Zn-Metalloproteases Angiotensin Converting Enzyme; ACE inhibitors for high blood pressure control -secretase in A production 11

12 Protease Case Study; - Secretases -secretase membrane-tethered metalloprotease -secretase membrane-anchored aspartyl protease -secretase oligomeric complex containing presenilins 1 and 2, aspartyl proteases Proteolysis of the Amyloid Precursor Protein Tandem action of - then -secretase yields A 1-40 or 1-42 while action of -secretase blocks A formation 12

13 -secretase catalyzes initial step in A formation, likely pharmacologic target. Xray structure allows structure-driven design of inhibitors; mechanism allows ts analog design for and -secretases 13

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15 Biological role for members of the -secretase family The identification of -secretase as an integral membrane protease puts this protease in a family that also cleave SREBP in sterol metabolism and Notch in developmental signal transduction pathways. Notch cleavage by - secretase releases the intracytoplasmic domain to go to the nucleus and function as a transcription factor. (Esler and Wolfe, Science 293, 1453, 2001) More general biological roles for -secretase protease family members include ectodomain shedding of membrane proteins and receptors such as TNF, the TNF p75 receptor, TGF, and L-selectin adhesion molecules 15

16 Caspase Cascades in Apoptotic Pathways: Metazoans eliminate cells that are damaged, mislocated, have become superfluous Activate an execution program of a cascade of controlled proteases, involving the Caspase family. Various stimuli activate the program Specific ligation of death receptors Ionizing radiation antineoplastic drugs Withdrawl of growth factors Convergence to a common execution program Signal at cell membrane Signal transduction via receptors with Death Domains and Death Effcetor Domains Protease Cascade with initiator caspases, amplifier Caspases, executioner Caspases that protelyze key intracellular protein substrates Caspase Cascade represents intracellular signaling pathways via proteolysis, with initiation, propagation, and termination phases. Initiation phase: Caspases activated hierarchically by protein-protein interactions to convert from single chain zymogens to two chain active proteases Propagation phase: Proteolysis of proteins at aspartyl residues. Some of these are pro forms of caspases, some are target proteins Termination Phase: Difficult to terminate the cascade once full amplification is underway. Low levels of active forms of Caspases can be blocked by intracellular antiprotease (IAP) proteins that act as tight-binding pseudosubstrates for Caspases, with picomolar to nanomolar Ki values.. Virally infected cells are loci for running apoptotic programs and viruses are known to produce caspoase inhibnitors to block the programmed cell death cascade: these include CrmA protein and p35. The p35 protein is cleaved at an internal asp residue but the two product fragments remain tightly bound and shut down the Caspase. The CrmA protein is also a substrate with selectivity for Caspase 1 and 8. Cells at rest: caspase are shut off, sitting around as inactive zymogens 16

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