Dr.Holzbaur 9.9.96 note: read lab handout before lab tomorrow. section B doesn't report to lab tomorrow - zoo trip. lab reports due the next week after the lab. A-Kerr is section B. General themes of biological regulation, continued. Synthesis of specific isozymes aka isoforms aka isoenzymes - closely related enzymes which activate the same reactions a bit differently. EXAMPLE: lactate dehydrogenase - catalyzes this rxn: pyruvate + NADH ----> lactate + NAD the reduction of pyruvate to lactate. LDH is a multisubunit enzyme that takes two forms - M muscle, H heart the subunits can combine as M4, M3H, M2H2, MH3, H4. M4 common in muscle, H form common in heart. M4 form has lower Km for pyruvate (eg, higher specificity for it) and also has a higher Vmax - therefore, this form of the enzyme is better at catalyzing this reaction, which is good in skeletal muscle. The H4 form has a lower affinity for pyruvate - doesn't bind it as well - higher Km, lower Vmax, so when H4 around, in heart, doesn't produce as much lactate, which is good, because don't want lactic acid building up in constantly working muscle. fetal form of Hb has higher affinity for O2 than that of adult Hb. That's how o2 transferred into fetal circulation. Glycogen storage disease type VII, in English Springer Spaniels. € chronic hemolytic disorder € exertional myopathy (causing exercise intolerance) The critical enzyme is € phosphofructokinase. in the sick dogs, this enzyme is only at 1-4% of normal activity. This enzyme has L,M,and C subunits, and in the sick dog there is no M type. The DNA which codes for this protein in the sick dog has a single point mutation which causes a truncated protein to form. Not much is lost from the protein - 40 AAs from the C terminal. But, the whole thing gets targeted for degradation because it isn't normal. M is the muscle form, and it isn't present in the muscle! VITAMINS AND COENZYMES: AA side chains offer flexibility in terms of structure, chemical properties, etc, but not enough to do everything cell needs to do. Many rxns require additional factors, or cofactors. € metal ions € organic type molecules - coenzymes these increase the range of functional groups available for catalysis, and serve as intermediary carriers for functional groups. When an enzyme requires a cofactor, the enzyme itself, plus the cofactor, is referred to as a holoenzyme. The enzyme itself which requires the cofactor is referred to as an apoenzyme. Some of these cofactors act like substrates, diffusing around; others are tightly bound to the enzymes, and are called "prosthetic groups" - includes both covalent and non-covalent tight links. VITAMINs are precursors to the coenzymes Some required metal ions for enzyme function: € zinc Zn required for alcohol dehydrogenase € Mg magnesium required for almost all ATPase € Fe Iron required for cytochromes (eg hemoglobin) COENZYMES: be aware of importance of carrier molecules and the groups they carry in activated form - table 17-2. ATP: used in the cell for a huge array of processes, used to donate phosphate groups to phosphorylate proteins, critical for DNA ad RNA structure, also a critical coenzyme involved in driving a lot of unfavorable enzymes. High concnetrations of atp are present in cells. In reactions involving ATP as a cofactor to drive an unfavorable rxn, mechanism is that the loss of a terminal phosphate group is SO favorable, with large free energy change O | O=P- | O will come off and add onto other molecule., and the former ATP molecule will pick up an H on the trailing O. Condensation/dehydration rxn A-H + B-OH -----> A-B B-OH + ATP ----> B-O-[P] + ADP transient A-H + B-O-[P]-----> A-B + Pi check this out in handout. so, the high energy intermediate B-O-[P[ is formed only transiently. [P] is a phosphate group. vitamins Niacin is a precursor for two coenzymes, NAD and NADP, which form NADH and NADPH. affinities vary a thousandfold. rxns which require these cofactors bind them very tightly and specifically, although a few will accept either factor. another related coenzyme is formed from the vitamin riboflavin. FMN and FAD are two major flavin nucleotides. Flavoenzymes bind these as prosthetic groups. The flavoenzymes are oxidation-reduction enzymes. niacin and riboflavin are water soluble vitamins, not stored well in mammals and birds, need to include them in diet, or are produced by microbes in the gut - especially in the ruminants. In cases of deficiency, different species respond differently, and different tissues differently too. riboflavin deficiency in young animals causes reduced growth rate, cataracts, curly-toe paralysis in the bird (myelin sheath degeneration). Hard to predict what form deficiency will take. Key biological role of these coenzymes is oxidation reduction reactions. PANTOTHENIC ACID acts with ß-mercaptoethylamine as coenzyme A- serves as a carrier of acyl groups in various rxns. deficiency can cause "goose stepping" from nerve problem. Thiamine: B1 needed for thiamin pyrophosphate, deficiency causes beribery in man and polyneuritis in birds. Tetrahydrofolate very important for synthesis of DNA....methotrexate is structural analogue of these enzymes which binds more tightly to an enzyme. Vitamin C, ascorbic acid. few animals get deficient in this vitamin (primates, guinea pigs, fruit bats, rainbow trout, silkworms) Dr holzbaur doesn't believe in megadoses of vit c having any useful effect. see table 8-3 for coenzymes which are synthesized from water soluble vtamins. table 11.11 is lipid soluble vitamins - these don't need to be in diet all the time, can store them in the body....note, can also get hypervitaminosis with these vitamins. Vitamin A/retinoic acid - deficiency leads to poor vision, but can also lead to many other dz states. Vitamin D -cholecalciferol - important in bone formation. can cause ricketts (deficiency). synthesized in skin in sunlight, dark animals can have problems, also inside animals. vitamin K required for synthesis of prothrombin which plays key role in blood clotting pathway. most animals get K from gut flora. Pigs sometimes eat feces to get vitamin K, so wire caging can cause deficiency; also cows fed hay stored too long can have deficiency, because sweet clover which spoils has dicumarol in it, and it inhibits the prothrombin synthesis reaction by competing w/vit K. "sweet clover disease".