Section 5. Enzymes, Equilibrium, Energy and the Sulfonamides

Section 5 Enzymes, Equilibrium, Energy and the Sulfonamides

Monday: ESKAPE handout describing them (Tiffany will provide). M-W Tie the metabolism back to the nutritional requirements and media choice, oxygen utilization. Mention liquid culture, growth curves perhaps in reference to MIC prev week? Simon: liq vs solid medium, growth rates. Wed lecture: brief sentence about phylogeny, close ESKAPE relative (genera share many of the same features/metabolic requirements, reflected in DNA similarity; virulence factors). If get a hit, then would follow up with the real pathogen. Chance for further research. Friday: sample exam question

Section 5 Learning Goals Be able to explain the mechanism of action of metabolic inhibitors at the molecular level Know the factors that determine rates of chemical reactions. Know the factors that determine equilibrium of chemical reactions. Explain how enzymes achieve substrate specificity Describe the molecular basis for antibiotic-target binding (covalent, non-covalent bond formation) Explain binding equilibrium as it pertains to antibiotic-target binding (and how it affects efficacy of an antibiotic) Describe how metabolic inhibitors achieve prokaryotic specificity.

Tortora p. 556 (10 th ed.) Antibiotic targets

Antibacterial metabolic inhibitors Sulfa drugs and trimethoprim are examples Exert effect by inhibiting key metabolic enzymes Folic acid derivatives are required for humans and for bacteria How do the metabolic inhibitor antibiotics achieve prokaryotic specificity?

Folic Acid is important for human health Intake from diet Derivatives are necessary for synthesis of DNA and amino acids Especially important for rapidly dividing cells Deficiency in pregnancy can lead to neural tube defects Folic acid (vitamin B 9 ) conversion in liver Dihydrofolic acid (DHF) Dihydrofolate reductase (DHFR) tetrahydrofolic acid (THF) Thymidine synthesis Amino acid synthesis

Bacteria synthesize their own folic acid derivatives Dihydropteroate diphosphate (DHPP) Dihydropteroate synthase (DHPS) + Dihydropteroic acid p-aminobenzoic acid (PABA) Folic acid (vitamin B 9 ) conversion in liver Dihydrofolic acid (DHF) Dihydrofolate reductase (DHFR) tetrahydrofolic acid (THF) Thymidine synthesis Amino acid synthesis

Metabolic inhibitor antibiotics interfere with function of critical enzymes in the pathway Dihydropteroate diphosphate (DHPP) Dihydropteroate synthase (DHPS) + X Dihydropteroic acid p-aminobenzoic acid (PABA) sulfonamides Folic acid (vitamin B 9 ) conversion in liver Dihydrofolic acid (DHF) Dihydrofolate reductase X trimethoprim (DHFR) tetrahydrofolic acid (THF) Thymidine synthesis Amino acid synthesis

Be able to explain the mechanism of action of metabolic inhibitors at the molecular level Requires knowledge of: Chemical reactions Products and reactants Gibbs free energy and ATP Equilibrium Enzymes and activation energy Relationship between concentration, rate, equilibrium and enzymes How enzymes achieve substrate specificity Binding kinetics Inhibition of enzyme activity

Chemical Reactions Collision theory probability of chemical reaction Velocity of reactants Concentration of reactants Temperature, pressure (# collisions and velocity) Enzymes tether reactants Orientation Higher effective concentration

Key Principle in Biology and Chemistry Equilibrium Most things are in equilibrium between two states An atom can bond with and dissociate from a carboxylic acid More likely to be bound at lower ph

Concentration influences rate of collision Heat increases kinetic energy of molecules. Therefore, it increases the frequency and velocity of collisions. What are the limitations to a cell using heat to increase the rate of enzymatic reactions?

Figure 5.6

Section 5 Learning Goals Be able to explain the mechanism of action of metabolic inhibitors at the molecular level ü Know the factors that determine rates of chemical reactions. ü Know the factors that determine equilibrium of chemical reactions. Explain how enzymes achieve substrate specificity Describe the molecular basis for antibiotic-target binding (covalent, non-covalent bond formation) Explain binding equilibrium as it pertains to antibiotic-target binding (and how it affects efficacy of an antibiotic) Describe how metabolic inhibitors achieve prokaryotic specificity.

Enzymes as Antibiotic Targets Enzymes are usually proteins Certain RNAs also has catalytic activity these are ribozymes Enzymes can be good targets for antibiotics since they naturally bind small molecules

Key Elements of Enzymes Collision theory Activation energy Co-factors used in many reactions Vitamins are often co-factors Metals are co-factors Co-factors transfer electrons readily

Figure 5.2

True or false? The free energy released will be greater if the reaction is catalyzed by an enzyme. The rate of the reaction increases in the presence of an enzyme. The activation energy required for a reaction to proceed decreased in the presence of enzyme.

Equilibrium of a reaction is affected by: A. Change in free energy between products and reactants B. Temperature Choose all that apply C. Concentration of reactants D. Activation energy required E. Rate at which the reaction proceeds F. Presence of enzyme

Rate of a reaction is affected by: A. Change in free energy between products and reactants B. Temperature Choose all that apply C. Concentration of reactants D. Presence of active enzyme

Favorable non-covalent bonding interactions increase molecular binding specificity Binding is transient

Sulfonamides mimic structure of PABA, tricking DHPS to catalyze the wrong reaction Dihydropteroate diphosphate (DHPP) Dihydropteroate synthase (DHPS) + p-aminobenzoic acid (PABA) Dihydropteroic acid

Sulfonamides mimic structure of PABA, tricking DHPS to catalyze the wrong reaction Dihydropteroate diphosphate (DHPP) Dihydropteroate synthase (DHPS) + p-aminobenzoic acid (PABA) sulfonamides Dihydropteroic acid Wrong product created

Sulfonamide structure resembles that of PABA Enzyme recognizes sulfonamide as PABA, adds it to DHPP Downstream reactions fail Dihydropteroate diphosphate (DHPP) p-aminobenzoic acid (PABA) Dihydropteroic acid sulfonamides

Enzyme Inhibitors Small molecules that exhibit non-covalent bonding to enzyme Bind to the active site (competitive inhibition) Bind to site other than active site alters conformation of active site (allosteric inhibition)

Trimethoprim is a competitive inhibitor of DHFR Dihydropteroate diphosphate (DHPP) Dihydropteroate synthase (DHPS) + p-aminobenzoic acid (PABA) sulfonamides Folic acid (vitamin B 9 ) conversion in liver Dihydropteroic acid Dihydrofolic acid (DHF) Dihydrofolate reductase X trimethoprim (DHFR) tetrahydrofolic acid (THF) Thymidine synthesis Amino acid synthesis

Efficacy of an inhibitor depends on its affinity for the target Binding is transient Binding affinity refers to how often a molecule is in the bound state vs. unbound state substrate A + inhibitor B on off AB bound enzyme

Binding affinity of antibiotic for substrate must be great enough to compete with binding of the normal substrate

Movie illustrating the dynamic and transient nature of binding http://www.youtube.com/watch? v=8xqtawerowm&feature=youtu.be Movie credit: life.uiuc.edu/emad/aac/ Copyright permissions needed From University of Illinois at Urbana-Champaign Theoretical and Computational Biophysics Group

Section 5 Learning Goals ü Be able to explain the mechanism of action of metabolic inhibitors at the molecular level ü Know the factors that determine rates of chemical reactions. ü Know the factors that determine equilibrium of chemical reactions. ü Explain how enzymes achieve substrate specificity ü Describe the molecular basis for antibiotic-target binding (covalent, non-covalent bond formation) ü Explain binding equilibrium as it pertains to antibiotic-target binding (and how it affects efficacy of an antibiotic) ü Describe how metabolic inhibitors achieve prokaryotic specificity.

Think-Pair-Share 1. How do the sulfonamides achieve prokaryotic specificity? 2. How does trimethoprim achieve prokaryotic specificity?

How do the sulfonamides achieve prokaryotic specificity?

Sulfonamides affect biochemical reaction not present in humans Dihydropteroate diphosphate (DHPP) Dihydropteroate synthase (DHPS) + p-aminobenzoic acid (PABA) sulfonamides Folic acid (vitamin B 9 ) Intake from diet human-specific conversion in liver Dihydropteroic acid Dihydrofolic acid (DHF) Dihydrofolate reductase X trimethoprim (DHFR) tetrahydrofolic acid (THF) Bacteria-specific Thymidine synthesis Amino acid synthesis

How does trimethoprim achieve prokaryotic specificity?

Trimethoprim has much lower binding affinity for the human version of DHFR Dihydropteroate diphosphate (DHPP) Dihydropteroate synthase (DHPS) + p-aminobenzoic acid (PABA) sulfonamides Folic acid (vitamin B 9 ) Intake from diet human-specific conversion in liver Dihydropteroic acid Dihydrofolic acid (DHF) Dihydrofolate reductase X trimethoprim (DHFR) tetrahydrofolic acid (THF) Bacteria-specific Thymidine synthesis Amino acid synthesis