BMS 100 Lecture 5: Proteins + Enzymes 1 PDF

Lecture 5: Proteins + Enzymes 1 In-class Dr. Rhea Hurnik BMS 100 Outline Proteins Classification Structure: Primary, secondary, tertiary, quaternary Denaturation Enzymes 1 Specificity Mechanisms Acid Base, Covalent catalysis Cofactors and Coenzymes Effect of temperatures and pH Regulation...

Lecture 5: Proteins + Enzymes 1 In-class Dr. Rhea Hurnik BMS 100 Outline Proteins Classification Structure: Primary, secondary, tertiary, quaternary Denaturation Enzymes 1 Specificity Mechanisms Acid Base, Covalent catalysis Cofactors and Coenzymes Effect of temperatures and pH Regulation Protein Structure • During pre-learning we look at the various levels of protein structure § Primary, secondary, tertiary, and quaternary • Let’s consider the loss of protein structure – ie disruption in folding/shape § This is called protein denaturation § Occurs when the bonds holding the protein together are disrupted. • What are some examples of these bonds? Protein denaturation: General • Bonds within proteins can be disrupted and the proteins denatured by using: § Strong acids or bases, or reducing agents: • Add or remove hydrogens § Organic solvents, detergents: • Disrupt hydrophobic, polar and charged interactions § Salts: • Disrupt polar and charged interactions § Heavy metal ions H2 N Salt bridge Protein Denaturation – Heavy Metals • Let’s consider how heavy metals will denature proteins § Ex: mercury (Hg+2) and lead (Pb+2) • Based on charge, what type of amino acid side chain do you think these heavy metals can bind to? § What type of interaction within a protein would therefore be disrupted? Protein Denaturation – Heavy Metals cont. • As well as binding to negatively charged groups, heavy metals can also bind to sulfhydryl (SH) groups § This alters the shape of the protein § This is the basis of “lead poisoning” • Pb+2 binds to the SH groups in two enzymes needed for the synthesis of Hb § Lack of Hb synthesis leads to the hallmark anemia associated with lead poisoning In class assignment time Enzymes • Enzymes outline: § Specificity § Mechanisms § Role of cofactors and coenzymes § Effect of temperatures or pH § Regulation New topic - Enzymes • Enzymes are an example of a type of globular protein. § Most biochemical reactions that occur in our body require enzymes in order to occur at a pace fast enough to support life. • We’ve already learned about several enzymes already! § Can you name one from glycolysis? Enzymes introduction • Enzymes = protein catalysts that speed up specific reactions and remained unchanged by the reaction § Enzymes speed up a reaction by lowering the activation energy of the reaction without changing: • The standard free energy (ΔG) of the reaction • The equilibrium of the reaction § They speed up a specific reaction by lowering the activation energy Ea Activation Energy (Ea) • The minimal amount of energy needed to make/break the bonds necessary for a reaction to occur § Also known biochemically as the free energy of activation (ΔG‡) § Sometimes defined as the amount of energy needed to reach the transition state (TS) • The transition state is the highest energy configuration formed when changing from reactants to products • Transient and not isolated Activation Energy (Ea) • To note: Free energy (𝚫G‡ ) Progress of reaction § The activation energy is lowered in the catalyzed reaction § No change in overall standard free energy of the equation (ΔG) Enzymes specificity • Enzyme molecules contain a special cleft called the active site § The active site forms by precise quaternary structure of the protein § Amino acids in the active site participate in substrate binding and catalysis Enzyme specificity • Enzymes are highly specific • Only a substrate of the correct size and shape can enter into the active site. § Once inside the site, the substrate binds the enzyme forming an enzyme-substrate (ES) complex § Binding of substrate is thought to induce a conformational change in shape of the enzyme • This is called the Induced fit model Enzyme mechanisms • Once a substrate binds to the active site, how exactly does it speed up a reaction? • The induced-fit between the correct substrate and enzyme allows for: § Electrostatic interactions to form between the two § The correct positioning of catalytic groups in the enzyme • Catalytic groups may speed up reactions in two main ways • Acid-base catalysis • Covalent catalysis Enzyme mechanisms: Acid-base effects • Addition or removal of a proton can make a substrate more reactive § The side chains of certain amino acids can add or remove H+’s by acting as general acids or bases • Which amino acid side chain do you think can function easily as an acid or a base? Enzyme mechanisms: Acid-base effects • Example Enzyme mechanisms: Covalent catalysis • A nucleophilic side group in the enzyme active site forms a temporary covalent bond with the substrate § Common nucleophilic side groups include: • Asp and Glu (R-COO-) • Ser (R-OH) and Cys (R-SH) § Serine and Cystein are only weakly nucleophilic, but their nucleophilicity is enhanced by the presence of other amino acids that can remove the H Enzyme mechanisms: Covalent catalysis • Example Cofactors and Coenzymes • Enzymes often have help from cofactors and coenzymes § Cofactors are typically metal cations • Mg2+, Zn2+ § We have already discussed the role of Mg2+ in several glycolytic enzymes involving ATP. What was the role of magnesium? Cofactors and Coenzymes • Enzymes often have help from cofactors and coenzymes § Cofactors are typically metal cations • Mg2+, Zn2+ § We have already discussed the role of Mg2+ in several glycolytic enzymes involving ATP. What was the role of magnesium? • Magnesium helped to position the ATP in the enzyme active site • Helped to stabilize the negative changes on the ATP Cofactors and Coenzymes • Enzymes often have help from cofactors and coenzymes § Coenzymes • Typically derived from vitamins • What are some examples of coenzymes we have learned so far? Cofactors and Coenzymes • Enzymes often have help from cofactors and coenzymes § Coenzymes • Typically derived from vitamins • What are some examples of coenzymes we have learned so far? § B3: NAD+ ßà NADH + H+ • Can accept or donate elections in redox reactions Cofactors and Coenzymes • In summary, cofactors and coenzymes can help enzymes speed up reactions in three main ways: § Can help position the substrate in the active site of the enzyme § Can stabilize negative charges on the substrate or the TS to make it easier for a nucleophilic attack to occur § Can accept/donate electrons in redox reactions Effect of temperature • The optimal temperature for an enzymes is usually the temperature of the organism § Do you think a fevers are good or bad? Effect of pH • Changing the pH can change the protonation state of the enzyme and/or the substrate § What types of bonds between E and S would potentially be disrupted by a change in pH? • H-bonds – for example: § If an H is removed, no H-bond can be formed § If an H is added, an H-bond might form that is not usually formed. • Electrostatic interactions - for example: § Adding an H can turn COO- into COOH § Removing an H can turn NH3+ into NH2 Effect of pH – Thinking question: • Consider the lysosome § What is the function of a lysosome? • Main site of intracellular enzymatic degradation for a wide range of molecules • At what pH do you suppose lysosomal enzymes are active at? (pH of ~7 or pH of ~45?) • What would happen to a lysosomal enzyme if it were release into the cytosol Lysosome Enzymes regulation • The activity of enzymes can be controlled in four main ways § 1. Genetic § 2. Covalent modification § 3. Allosteric regulation § 4. Compartmentalization Enzymes regulation: Genetic • Enzymes transcription can be induced or repressed based on the needs of the cell § In times of needs enzyme transcription and translation will be induced § In times of excess enzyme transcription and translation will be repressed • We have already discussed several ways in which this can happen. § What were some examples? Enzymes regulation: Genetic • Let’s look at an example: Regular consumption of a meals rich in carbohydrates High insulin Increased transcription of genes for glucokinase, PFK-1, and pyruvate kinase Increased translation Higher amount of glucokinase, PFK-1, and pyruvate kinase in the cytosol More efficient conversion of glucose to pyruvate Enzyme regulation: Covalent modification • Involves altering the structure of an enzyme (or “proenzyme”) by making or breaking covalent bonds § There are two types of covalent modification: • Reversible • Irreversible Enzyme regulation: Covalent modification • Reversible covalent modification § Involves addition or removal of a group to the enzyme that causes it to convert to its active or inactive form • What is a common group that can do this? § Does addition of this group activate or inactivate the enzyme? • Other common groups that can be added to or removed enzymes include methyl and acetyl groups Enzyme regulation: Covalent modification • Example – glycogen metabolism § The main regulated enzyme in glycogenesis is deactivated by phosphorylation while the main enzyme in glycogenolysis is activated by phosphorylation. • Phosphorylation is catalyzed initially by the same protein, protein kinase A (PKA) • This prevents both pathways from running at the same time Enzyme regulation: Covalent modification activated Inhibited • Example – glycogen metabolism Protein kinase A ATP Protein kinase A ADP ATP Glycogen phosphorylase kinase Glycogen phosphorylase kinase ATP Glycogen phosphorylase Glycogen synthase P ADP Glycogen synthase ADP Glycogen phosphorylase Glycogenolysis P No glycogenesis P Enzyme regulation: Covalent modification activated Inhibited • Example – glycogen metabolism continued Protein kinase A ATP Protein kinase A ADP ATP Glycogen phosphorylase kinase Glycogen phosphorylase kinase ATP Glycogen phosphorylase Glycogen synthase P ADP Glycogen synthase ADP Glycogen phosphorylase Glycogenolysis P No glycogenesis P Enzyme regulation: Covalent modification • Irreversible covalent modification § Involves cleavage of peptide bonds in proenzymes or zymogens § Makes sure the enzyme is not used until it is in the correct location and until it is needed Enzyme regulation: Allosteric modification • Allosteric modification of allosteric enzymes § Binding to enzyme’s allosteric site changes the conformation and activity of the enzyme • Changes the binding affinity of the substrate at the active site § More to come in Enzymes II § Commonly used to control regulatory enzymes Enzyme regulation: Allosteric modification • Allosteric enzymes have more than one subunit § Allosteric site is on one subunit, active site on another § The binding of an effector molecule to an allosteric enzyme can either: • Increase binding of the substrate to the enzyme § Effector = activator • Decrease binding of the substrate to the enzyme § Effector = inhibitor Enzyme regulation: Allosteric modification • Consider the glycolytic enzyme phosphofructokinase-1 (PFK-1): • What reaction is catalyzed by PFK-1? It is reversible or irreversible? § Phosphofructokinase-1 is inhibited allosterically by high levels of ATP • Can you think of why this might be the case? § Phosphofructokinase-1 in activated allosterically by high levels of AMP • Can you think of why this might be the case? Enzyme regulation: Compartmentalization • Compartmentalization of enzymes via membranebound organelles allows for regulation by: § 1. Separation of enzymes from opposing pathways into different cellular compartments, and selective transportation of substrates • Can you think of metabolic pathways that are compartmentalized in different areas of the cell? Enzyme regulation: Compartmentalization • Compartmentalization of enzymes via membranebound organelles allows for regulation by: § 2. Creation of unique microenvironments • Lysosomal enzymes function at a pH around 4.5-5, while most other cellular enzymes function at a pH around 7.