BIOL 430 Notes - Summary Lehninger Principles of Biochemistry PDF

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Summaries of Ch.5 and Ch. 6....

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Chapter 5: Protein Function IgG binding to a pathogen activates leukocytes (ex. macrophages) that destroy the invader. Receptors on the macrophage bind the Fc region of IgG. When the Fc receptors binds an antibody-antigen complex, the macrophage engulfs the complex by phagocytosis. IgE → allergic rhinitis Antibodies Bind Tightly and Specifically to Antigen The binding specificity of an antibody is determined by the amino acids in the variable domains of the light and heavy chains. Residues lining the antigen-binding site are hypervariable. Antibody-antigen binding = induced fit Low Kd (high affinity, tight binding) reflects energy derived from forces that stabilize binding (hydrophobic effect, ionic interactions, H-bonding, van der Waals interactions) The Antibody-Antigen Interaction is the Basis for a Variety of Important Analytical Procedures Polyclonal antibodies are produced by many different B lymphocytes responding to one antigen. Polyclonal preparations thus have a mixture of antibodies that recognize different parts of the protein. Monoclonal antibodies are synthesized by a population of identical B cells. All of these recognize the same epitope. Practically, a selected antibody can be covalently attached to a resin ad used in a chromatography column. When a mixture of proteins is added to the column, the antibody specifically binds its target protein and retains it on the column while other proteins elute. The target protein can then be eluted from the resin. ELISA Western Blot 5.3 Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors The Major Proteins of Muscle Are Myosin and Actin Myosin has two heavy chains and four light chains. Each heavy chain has a large globular domain containing a site where ATP is hydrolyzed. In muscles, myosin molecules aggregate to form thick filaments. These are the core of the contractile unit. G-actin (monomer) associated to form F-actin (polymer). F-actin with troponin and tropomyosin make up the thin filament. Each actin monomer in the thin filament can bind tightly and specifically to one myosin head group. Additional Proteins Organize the Thin and Thick Filaments into Ordered Structures The entire contractile unit is the sarcomere. Myosin Thick Filaments Slide along Actin Thin Filaments

Actomyosin cycle 1. ATP binds to myosin head, causing dissociation from actin. 2. As tightly bound ATP is hydrolyzed, a conformational change occurs. ADP Pi remain associated with the myosin head. 3. Myosin head attaches to actin filament, causing release of Pi. 4. Pi release triggers a “power stroke,” a conformational change in the myosin head that moves actin and myosin filaments relative to each other. ADP is released in the process. Tropomyosin and troponin mediate regulation of actin-myosin interaction. Tropomyosin binds to thin filament and blocks attachment sites for myosin heads. Calcium released from sarcoplasmic reticulum via a nerve impulse binds to troponin and causes a conformational change that exposes actin’s myosin-binding sites. This is followed by the actomyosin cycle. Myosin is both an actin-binding protein and an ATPase - an enzyme. Chapter 6: Enzymes 6.1 An Introduction to Enzymes Most Enzymes Are Proteins Other than catalytic RNA, all enzymes are proteins. All levels of protein structure are essential to catalytic activity. Some enzymes require a cofactor - an inorganic metal ion or an organic molecule (coenzyme). Coenzymes act as transient carriers of specific functional groups. (vitamin-derived) A cofactor that is tightly/covalently bound to the enzyme is a prosthetic group. A complete, active enzyme with cofactor(s) bound is a holoenzyme. The protein part is the apoenzyme/apoprotein. Enzymes Are Classified by the Reactions They Catalyze LILHOT Ligases: formation of C-S, C-C. C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor Isomerases: transfer of groups within molecules to yield isomeric forms Lyases: cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds Hydrolases: hydrolysis reactions (transfer of functional groups to water) Oxidoreductases: transfer of electrons (hydride ions or H atoms) Transferases: group transfer reactions 6.2 How Enzymes Work An enzyme provides a specific environment (active site) in which a given reaction can occur more rapidly. Enzymes Affect Reaction Rates, Not Equilibria E+S ←→ ES ←→ EP ←→ E+P

ES and EP are transient complexes. The starting point for either the forward or reverse reaction is called the ground state, the contribution to the free energy of the system by an average molecule (S or P). The rate of a reaction is dependent on the activation energy. At the transition state, decay to the S or the P state is equally probable. A transition state is unstable and is NOT a reaction intermediate like ES and EP. The ES and EP complexes are the valleys in the reaction coordinate diagram. The transition states are the hill. A higher activation energy corresponds to a slower reaction. Enzymes catalyze BIDIRECTIONAL reactions. The enzyme is unaffected in the process, and the equilibrium point is unaffected. In a multistep reaction, the step with the highest activation energy (slowest step) is the ratelimiting step. Reaction Rates and Equilibria Have Precise Thermodynamic Definitions �G’°= -RTlnK’eq A large-� G’° reflects a favorable reaction equilibrium. (this has nothing to do with rate) For the unimolecular reaction S→ P, the rate of the reaction (representing the amount of S that reacts per unit time) is expressed by a rate equation: V=k[S] A Few Principles Explain the Catalytic Power and Specificity of Enzymes Rearrangement of covalent bonds during an enzyme-catalyzed reaction 1. Catalytic functional groups on an enzyme may form a transient covalent bond with a substrate and active it for reaction, or a group may be transiently transferred from the substrate to the enzyme. Covalent interactions lower the activation energy by providing an alternate, lower-energy reaction. 2. Non-covalent interactions between enzyme and substrate stabilize protein structure and protein-protein interactions. Formation of each weak interaction in the ES complex is accompanied by release of a small amount of energy that stabilizes the interaction. (binding energy, �G’B) Weak Interactions between Enzyme and Substrate Are Optimized in the Transition State In order to catalyze reactions, an enzyme must be complementary to the reaction transition state. Adequate binding is accomplished most readily by positioning a substrate in the active site, where it is effectively removed from water. Binding Energy Contributes to Reaction Specificity and Catalysis Specificity is derived from the formation of many weak interactions between the enzyme and its specific substrate. Binding energy constrains the substrates in the proper orientation to react, reducing entropy. Formation of weak bonds between substrate and enzyme results in desolvation of the

substrate by replacing most or all of the hydrogen bonds between the substrate and water. Specific Catalytic Groups Contribute To Catalysis 1. General acid-base catalysis Charged intermediates can often be stabilized by the transfer of protons to form a species that breaks down more readily to products. These protons are transferred between a enzyme and a substrate or intermediate. Catalysis that only use H+ and OHpresent in water is specific acid-base catalysis. In general acid-base catalysis, proton transfers are mediated by weak acids and bases other than water. (Water may not be available in an active site). 2. Covalent catalysis A transient covalent bond is formed between the enzyme and the substrate. Several amino acid side chains and some functional groups can act as nucleophiles. 3. Metal ion catalysis Mediate oxidation-reduction reactions by reversible changes in oxidation state. 6.3 Enzyme Kinetics as an Approach to Understanding Mechanism Substrate Concentration Affects the Rate of Enzyme-Catalyzed Reactions At relatively low [S], V0 (initial velocity) increases almost linearly with increase in [S]. At higher [S], V0 increases by smaller and smaller amounts until it plateaus. This plateau region is close to the maximum velocity, Vmax. The overall rate must be proportional to the concentration of the species that reacts in the second (slower) step - ES. At Vmax, virtually all the enzyme is present as the ES complex and [E] is vanishingly small. The enzyme is saturated with substrate at this time, so further increases in [S] have no effect on rate. After the ES complex breaks down to yield the product, the enzyme is free to catalyze again. Saturation = plateau When the enzymes if first mixed with a high level of substrate, there is an initial pre-steady state when [ES] builds up. Then steady state, where [ES] remains approx. constant over time. The measured Vo reflects the steady state.

The Relationship between Substrate Concentration and Reaction Rate Can Be Expressed Quantitatively V0=Vmax[S]/Km+[S] Steady-state assumption: the rate of formation of ES is equal to the rate of its breakdown Km=[S] when V0=1/2Vmax

Kinetic Parameters Are Used to Compare Enzyme Activities All enzymes that exhibit a hyperbolic dependence of V0 on [S] are said to follow MichaelisMenten kinetics. Interpreting Vmax and Km kcat is a more general rate constant that describes the limiting rate of any enzyme-catalyzed reaction at saturation. It is a first order rate constant (s-1), and is also called the turnover number. It is equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate. kcat=Vmax/[Et] V0=kcat[Et][S]/Km+[S] Comparing Catalytic Mechanism and Efficiencies The ratio kcat/Km is the specificity constant. Many Enzymes Catalyze Reactions with Two or More Substrates Ex. hexokinase catalyzes a reaction between ATP and glucose that yields ADP and G6P. In some cases, both substrates are bound to the enzyme concurrently at some point, forming a noncovalent ternary complex. In other cases, the first substrate is converted to product and dissociates before the second substrate binds (Ping-Pong mechanism). Enzyme Activity Depends on pH Enzymes have an optimal pH range where their activity is maximal activity. A change in activity near pH 7 indicates titration of a His residue. Pre-Steady State Kinetics Can Provide Evidence for Specific Reaction Steps The observed rate of product formation slows to the steady state as the bound product is slowly released. Each enzymatic turnover after the first one must proceed through the slow product release step. The observation of a burst indicates that a rate-limiting step occurs after formation of the product being monitored. Enzymes Are Subject to Reversible or Irreversible Inhibition Reversible Inhibition Competitive inhibition→ same Vmax, higher Km Uncompetitive inhibition→ decrease in Vmax and Km Mixed inhibition→ decrease in Vmax, Km up or down Noncompetitive→ decrease in Vmax, same Km Uncompetitive and mixed inhibition are observed only for enzymes with two or more substrates. Irreversible Inhibition Irreversible inhibitors bind covalently with or destroy a functional group on the enzyme that is essential for its activity, or they form a highly stable noncovalent association.

Suicide inactivators combine irreversibly with the enzyme. Transition-state analogs 6.4 Examples of Enzymatic Reactions The Chymotrypsin Mechanism Involves Acylation and Deacylation of a Ser Residue Chymotrypsin is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. It is specific for peptide bonds adjacent to aromatic amino acid residues. Chymotrypsin uses both general acid-base catalysis and covalent catalysis. A transient covalent acyl-enzyme intermediate is formed. 1. Acylation phase a. Peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme 2. Deacylation phase a. The ester linkage is hydrolyzed and the nonacylated enzyme regenerated. Chymotrypsin → ES complex → short-lived intermediate (acylation) → product 1 and acyl-enzyme intermediate → acyl-enzyme intermediate, water added → short-lived intermediate (deacylation) → enzyme-product 2 complex → chymotrypsin and product 2 An Understanding of Protease Mechanisms Leads to New Treatments for HIV Infections HIV protease, an aspartyl protease, is most efficient at cleaving peptide bonds between Phe and Pro residues. Mechanism: 1. Aided by general base catalysis, water attacks the carbonyl carbon, generating a tetrahedral intermediate stabilized by hydrogen bonding. 2. The tetrahedral intermediate collapses; the amino acid leaving group is protonated as it is expelled. HIV protease inhibitors act as transition-state analogs. Hexokinase Undergoes Induced Fit on Substrate Binding Hexokinase is a bisubstrate enzyme that catalyzes the reaction of glucose and ATP to G6P and ADP. ATP and ADP always bind to enzymes as a complex with Mg2+. Specificity is observed not in the formation of the ES, but in the relative rates of subsequent catalytic steps. Reaction rates increase greatly in the presence of a substrate (glucose) that can be phosphorylated. The active site amino acid residues participate in general acid-base catalysis and transitionstate stabilization. The Enolase Reaction Mechanism Requires Metal Ions Enolase catalyzes the reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate. 1. Lys abstracts a proton by general base catalysis. Two Mg2+ ions stabilize the resulting enolate intermediate. 2. Glu facilitates elimination of the -OH group by general acid catalysis.

Lysozyme Uses Two Successive Nucleophilic Displacement Reactions Substrate: peptidoglycan The key catalytic residues are Glu and Asp. An Understanding of Enzyme Mechanism Produces Useful Antibiotics Penicillin is an irreversible inhibitor of transpeptidase (formation of covalent complex). Beta-lactamases are enzymes that cleave beta-lactam antibiotics, rendering them inactive. (leads to bacterial resistance). 6.5 Regulatory Enzymes Allosteric enzymes reversibly and covalently bind regulatory compounds called allosteric modulators (small metabolites or cofactors). Other enzymes are regulated by reversible covalent modification. Both tend to be multisubunit proteins. Some enzymes are stimulated/inhibited when they are bound by regulatory proteins. Others are activated by proteolytic cleavage (irreversible). Allosteric Enzymes Undergo Conformational Changes in Response to Modulator Binding Often the modulator is the substrate itself (homotropic). Binding of the substrate causes conformational changes that affect the subsequent activity of other sites on the protein. Just as an enzyme’s active site is specific for its substrate, each regulatory site on an allosteric enzyme is specific for its modulator. In homotropic enzymes, the active site and regulatory site are the same. Allosteric enzymes are typically larger and more complex than others, with multiple subunits. Ex. aspartate transcarbamolyase (ATCase) - both homotopic and heterotopic, which catalyzes a step in synthesis of pyrimidine nucleotides. The regulatory subunits of ATCase have binding sites for ATP (positive regulator) and CTP (negative regulator). CTP is one of the end products of the pathway. ATP feeds into RNA transcription and DNA replication.

The Kinetic Properties of Allosteric Enzymes Diverge from Michaelis-Menten Behavior Sigmoidal curve, no Km Some Enzymes Are Regulated by Reversible Covalent Modification Introduction of a charge can alter the local properties of the enzyme and cause a conformational change. Phosphorylation, methylation, adenylylation, etc. Phosphoryl Groups Affect the Structure and Catalytic Activity of Enzymes

Protein kinases attach phosphate groups to specific amino acid residues of a protein. Ser, Thr, and Tyr can be phosphorylated. This introduces a bulky, charged group into a region that was only moderately polar. Protein phosphatases remove phosphate groups. Ex. phosphorylation kinase catalyzes phosphorylase b → phosphorylase a. Multiple Phosphorylations Allow Exquisite Regulatory Control Some proteins have consensus sequences recognized by multiple different protein kinases, each of which was phosphorylate it. Other proteins exhibit hierarchical phosphorylation: a certain residue can be phosphorylated only if a neighboring residue has already been phosphorylated. Some Enzymes and Other Proteins Are Regulated by Proteolytic Cleavage of an Enzyme Precursor For some enzymes, an inactive precursor called a zymogen is cleaved to form the active enzyme. Ex. chymotrypsinogen and trypsinogen. Specific cleavage causes conformational changes that expose the enzyme active site. This type of activation is irreversible. Proteases are inactivated by inhibitor proteins that bind very tightly to the active site. Proproteins/proenzymes are also activated by proteolysis. A Cascade of Proteolytically Activated Zymogens Leads to Blood Coagulation Fibrin is derived from a zymogen fibrinogen. The formation of a blood clot is an example of a regulatory cascade. Fibrinogen is a dimer of heterotrimers with three different types of subunits. It is converted to fibrin and thereby activated for blood clotting by proteolytic cleavage. Peptide removal is catalyzed by the serine protease thrombin. Fibrin polymerizes into a gel-like matrix to form a soft clot. Covalent cross-links between associated fibrins turn the soft clot into a hard clot. Some Regulatory Enzymes Use Several Regulatory Mechanisms Glycogen phosphorylase is regulated by covalent modification primarily, but also by allosteric binding of AMP. AMP is an activator of phosphorylase b, and G6P and ATP are inhibitor. The phosphorylases are also regulated by the levels of hormones that regulate blood sugar....