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...

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 H2N 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 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 Ea of the reaction without changing: • The standard free energy (ΔG) of the reaction • The equilibrium of the reaction 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+ 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 Cysteine 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: 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? ▪ B3: NAD+ 🡪🡪 NADH + H+ • Can accept or donate electrons 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 ~4.5?) • 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 The addition or removal of a group to the enzyme that causes it to convert to its active or inactive form. • 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 de-activated 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 inhibited active • 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): 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 membrane-bound organelles allows for regulation by: 1. Separation of enzymes from opposing pathways into different cellular compartments, and selective transportation of substrates 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.