Biochem Exam 2 PDF

18. Protein Functions: Catalysis Recommended reading: Chapter 6 (Lehninger Principles of Biochemistry. Nelson & Cox-7th Edition) Course Proficiencies covered: 1. Describe the relationships between informational, structural, and catalytic molecules in living organisms 2. Describe the fun...

18. Protein Functions: Catalysis Recommended reading: Chapter 6 (Lehninger Principles of Biochemistry. Nelson & Cox-7th Edition) Course Proficiencies covered: 1. Describe the relationships between informational, structural, and catalytic molecules in living organisms 2. Describe the functions and relationships of enzymes, coenzymes and cofactors Jayshree Mishra; PhD Office: Rm. 213 Poll Everywhere login: mishra  Learning goals: Describe the physiological significance of enzymes and its components. Describe the chemical mechanisms of catalysis Describe the regulation of enzyme activity Describe enzyme kinetics and inhibitions Key concept: Enzymes, the extremely specific biological catalysts, regulate the biochemical reactions at body temperature and pH without which life would be impossible. Enzymes?  What Are Enzymes? Enzymes are biological catalysts. increase reaction rates (105 to 1017) without (a) being used up or (b) affecting equilibrium. Most enzymes are proteins in proper configuration. However, some RNA (ribozymes and ribosomal RNA) also catalyze reactions. Some enzymes require a cofactor – An ion such as Fe++, Mg++ or Zn++ – An organic or organometallic coenzyme (e.g. heme, or NADP) – Holoenzyme is with the cofactor, apoenzyme is without the cofactor – Some cofactors are tightly bound (called prosthetic groups), others are loosely bound 1. Name one function of ribozyme? Enzyme Name (substrate/function)  Why do we need to understand the enzyme mechanism? -Penicillin -Amoxicillin and Clauvulanic acid -Antiviral therapy How enzymes work?  Enzymatic Catalysis Enzymes do not affect equilibrium (Keq). Which means that enzymes cannot affect the overall ΔG free energy of the reaction. Slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction. Enzymes increase reaction rates by decreasing ΔG‡.  Enzymes Decrease ΔG‡ ΔG ΔG = ΔH – TΔS  How do Enzymes Lower G Enzymes organize reactive groups into close proximity and proper orientation. Uncatalyzed bimolecular reactions Two free reactants single restricted transition state conversion is energetically unfavorable. Uncatalyzed unimolecular reactions Flexible reactant rigid transition state conversion is energetically unfavorable for flexible reactants. Catalyzed reactions The enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complex. The energy cost is paid during binding.  How do Enzymes Lower G Enzymes bind best at the transition states. The idea was proposed by Linus Pauling in 1946. – Enzyme active sites are complimentary to the transition state of the reaction. – Enzymes bind transition states better than substrates. – Stronger/additional interactions with the transition state as compared with the ground state lower the activation barrier. Cofactors and Associated Enzymes  Some Inorganic Ions That Serve as TABLE 6-1 Cofactors for Enzymes Ions Enzymes Cu2+ Cytochrome oxidase Fe2+ or Fe3+ Cytochrome oxidase, catalase, peroxidase K+ Pyruvate kinase Hexokinase, glucose 6-phosphatase, Mg2+ pyruvate kinase Mn2+ Arginase, ribonucleotide reductase Mo Dinitrogenase Ni2+ Urease Carbonic anhydrase, alcohol dehydrogenase, Zn2+ carboxypeptidases A and B 2. Name a cofactor that is also a part of skeletal system?  Coenzymes and Dietary precursors Some Coenzymes That Serve as Transient Carriers of Specific Atoms or TABLE 6-2 Functional Groups Coenzyme Examples of chemical groups Dietary precursor in mammals transferred Biocytin CO2 Biotin Coenzyme A Acyl groups Pantothenic acid B5 /other compounds 5'-Deoxyadenosylcobalamin H atoms and alkyl groups Vitamin B12 (coenzyme B12) Flavin adenine Electrons Riboflavin (vitamin B2) dinucleotide Lipoate Electrons and acyl groups Not required in diet Nicotinamide adenine Hydride ion (:H–) Nicotinic acid (niacin) B3 dinucleotide Pyridoxal phosphate Amino groups Pyridoxine (vitamin B6) Tetrahydrofolate One-carbon groups Folate (vitamin B9) Thiamine pyrophosphate Aldehydes Thiamine (vitamin B1) Note: The structures and modes of action of these coenzymes are described in Part II.  Classification of Enzymes TABLE 6-3 International Classification of Enzymes Class no. Class name Type of reaction catalyzed 1 Oxidoreductases Transfer of electrons (hydride ions or H atoms) 2 Transferases Group transfer reactions Hydrolysis reactions (transfer of functional 3 Hydrolases groups to water) Cleavage of C—C, C—O, C—N, or other 4 Lyases bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds Transfer of groups within molecules to yield 5 Isomerases isomeric forms Formation of C—C, C—S, C—O, and C—N 6 Ligases bonds by condensation reactions coupled to cleavage of ATP or similar cofactor 3. Is H- an electrophile or a nucleophile? Kinetics for enzymes  What Is Enzyme Kinetics? Kinetics is the study of the rate at which compounds react. The rate of enzymatic reaction is affected by: – enzyme – substrate – effectors – temperature  Why Study Enzyme Kinetics? Quantitative description of biocatalysis. Understand catalytic mechanism Find effective inhibitors Understand regulation of activity Effect of Substrate Concentration Vmax [ S ] v * Km  S How will the graph look like if you vary only [E]? Determination of Kinetic Parameters A nonlinear Michaelis-Menten plot should be used to calculate parameters Km and Vmax. A linearized double-reciprocal plot is good for analysis of two-substrate data or inhibition. Saturation Kinetics: at High [S], Velocity Is not Proportional to [S]  Chymotrypsin: a serine protease During digestion, dietary proteins must be broken down into small peptides by proteases. Chymotrypsin is one of several proteases that cuts peptides at specific locations on the peptide backbone. This protease is able to cleave the peptide bond adjacent to aromatic amino acids. Chymotrypsin cuts this bond. 4. For what medical condition is the chymotrypsin used? Chymotrypsin Mechanism Step 1: Substrate Binding Chymotrypsin Mechanism Step 2: Nucleophilic Attack Chymotrypsin Mechanism Step 3: Substrate Cleavage Chymotrypsin Mechanism Step 4: Water Comes In Chymotrypsin Mechanism Step 5: Water Attacks Chymotrypsin Mechanism Step 6: Break-off from the Enzyme Chymotrypsin Mechanism Step 7: Product Dissociates At this time you should know What are enzymes and how do they work Cofactor, coenzyme, holoenzyme, apoenzyme, prosthetic group, substrate, and active site. Coenzymes (e.g. CoA, FAD, NAD, and THF), the chemical groups it transfers, and the dietary precursor. Types of general catalytic mechanisms. The catalysis and the key amino acids that participate in specific catalytic reaction (e.g. chymotrypsin catalysis) Allosteric regulation of enzyme  Enzyme Activity Can Be Regulated Regulation can be: – noncovalent modification (allosteric) – covalent modification – irreversible – reversible  Noncovalent Modification: Allosteric Regulators Allosteric effectors or modulators are generally small chemicals. Allosteric effectors can be positive, or improve enzymatic catalysis. Allosteric effectors can be negative, or reduce enzymatic catalysis.  Noncovalent Modification: Allosteric Regulators The kinetics of allosteric regulators differ from Michaelis-Menten kinetics. Substrate also serves as a positive (stimulatory) synthesis ATP CTP ATCase catalyzes the condensation of aspartate and carbamoyl phosphate to form N-carbamoylaspartate, in pyrimidine Enzyme activity depends on pH  Enzyme Inhibition Inhibitors are compounds that decrease an enzyme’s activity. Irreversible inhibitors (inactivators) react with the enzyme. One inhibitor molecule can permanently shut off one enzyme molecule. They are often powerful toxins but also may be used as drugs. Bacterial ADP-ribosylating exotoxins (bAREs) covalently transfer an ADP- Ribose moiety of NAD+ to target proteins of infected eukaryotes. Read about bAREs in infection: cholera toxin enterotoxin; Exotoxin A; Pertussis toxin; C3 toxin; and Diphtheria toxin Reversible inhibitors bind to and can dissociate from the enzyme. They are often structural analogs of substrates or products. They are often used as drugs to slow down a specific enzyme. Reversible inhibitor can bind to: the free enzyme and prevent the binding of the substrate. (Km or Vmax)? the enzyme-substrate complex and prevent the reaction. (Km or Vmax)?