VMED 5110

Learner Objectives for Dr. Moroney's Lectures

Myoglobin and hemoglobin: Know myoglobin is a single subunit and reversibly binds O₂. Define oxymyoglobin, deoxymyoglobin, and metmyoglobin and hemoglobins; methemoglobinemia due to nitrate/nitrite (cattle) and acetaminophen (cats). Hemoglobin is a tetramer with 4 O₂ binding sites. Be able to identify oxygen-binding curves for myoglobin and hemoglobin and know the significance of the sigmoidal shape of the hemoglobin curve; T state and R state. Know BPG, H⁺, and CO₂ as allosteric effectors of hemoglobin and their effect on oxygen binding; know the effect of BPG on O₂ delivery to the fetus in humans, dogs, horses and pigs; know the problems of designing a hemoglobin-based blood substitute.

Enzymes:  How do enzymes catalyze reactions?  RNA enzymes. Transition state analogs are good enzyme inhibitors; antibodies as enzymes; how does pH affect enzyme activity? Michaelis-Menten equation (hyperbolic kinetics), Lineweaver-Burk plot (1/V vs. 1/[S], Km, Vmax).  Regulation of enzyme activity:  allosteric enzymes (sigmoidal kinetics); isozymes; proteolytic activation; covalent modification:  phosphorylation (ser, thr, tyr) and phosphatases, erb-B oncogene.

Clinical uses of enzymes:  The substrate concentration in a clinical assay is set at 5 to 10 times the K_m so that the rate of the reaction is proportional to the total enzyme concentration (it will be at V_max , which is proportional to the total enzyme concentration). Thrombin time, prothrombin time, and activated partial prothrombin time:  how you start the assay and what serum enzymes or proteins they measure (just the info that was on the slide).  ALP, ALT, AST, CK, GDH, gGT:  know the names and abbreviations and the conditions they are used to diagnose.  Lactate dehydrogenase isozymes (different forms of an enzyme that catalyze the same activity):  heart has H₄, which converts lactate to pyruvate; skeletal muscle has M₄, which converts pyruvate to lactate.  Streptokinase activates plasminogen in serum and bound to fibrin; tissue plasminogen activator activates plasminogen bound to fibrin; both act to dissolve clots.

Enzyme inhibition:  characteristics of competitive inhibition (inhibitor usually resembles substrate (warfarin looks like vitamin K), V_m is the same as uninhibited and K_m increases, what a Lineweaver-Burk plot for competitive inhibition looks like); irreversible inhibition, suicide inhibitors; sarin, malathion, and carbaryl are irreversible inhibitors of acetylcholinesterase, the insecticides are less toxic because they hydrolyze eventually; therapeutic inhibitors:  exploiting differences in biochemical pathways between bacteria, fungi, and animal hosts:  sulfonamides inhibit folate synthesis; b-lactam antibiotics (eg., amoxicillin) inhibit bacterial cell wall synthesis, some bacteria have plasmid-derived b-lactamases that confer drug resistance, clavulanate inhibits b-lactamase and is given in conjunction with amoxicillin to prevent resistance; 5-fluorocytosine can be used to treat fungal infections because fungi, but not animal cells, convert 5-fluorocytosine to 5-fluorouracil, which is converted to 5-fluoro-dUMP, which is a suicide inhibitor of thymidylate synthase.  Ethanol can be used to treat ethylene glycol intoxication because it is a competitive inhibitor of liver alcohol dehydrogenase.  Aspirin and acetaminophen are inhibitors of prostaglandin synthase (also known as COX-1 and COX-2); know the pathways for elimination of acetaminophen, and that CYP2E1 (a cytochrome P450) converts it to a toxic metabolite that must be conjugated with glutathione or it will kill cells.