The structure of ATP

2. The structure of ATP • Each ATP molecule has three subunits: • -Ribose sugar • -Adenine • -Triphosphate group formed by the linkage of three phosphate groups (PO4). How ATP stores, transfers, and releases energy • ATP stores energy released from other exergonic reactions in the covalent bonds that link the phosphate groups in ATP, called phosphoanhydride bonds. • The phosphoanhydride bonds are fairly unstable and require only small amount of free energy to break it, a process called hydrolysis. • The hydrolysis of terminal phosphoanhydride bond releases stored energy.

3. ATP is the Energy Currency of Cells • ATP “couples” energy-releasing (exergonic) and energy-storing (endergonic) enzymatic reactions in cells. • ATP, ADP, and phosphate are continually being recycled within living cells as cells use group transfer to capture, carry, and release energy.

4. Group Transfer by Phosphorylation • The stored energy in ATP is easily transferred, along with the PO4 group, to another molecules in a process called phosphorylation. • ATP has high group transfer potential. ATP loses stored energy due to hydrolysis to produce ADP and phosphate group. Compound A gains more energy due to phosphorylation.

5. The Chicken and the Egg Evolution Which came first proteins or DNA? Without DNA there is no way to direct protein synthesis, but DNA can not synthesize itself. In the first living organism it is believed that RNA (or even a precursor of RNA) was both the genetic information and the catalysts (a self-replicating RNA). How many reactions did these RNA organisms catalyze? It is believed that they had a diverse biochemistry. It is believed they used all of the base containing cofactors and molecules: NAD, NADP, CoA, and etc. The base was a “handle” for these ribozymes. If the organisms were that complex, they almost certainly had a membrane. It makes no sense to produce NADH or NAD if it is just going to float way.

6. Ribozymes Relatively new discoveries • Ribozymes - segments of RNA that display enzyme activity in the absence of protein • Examples: RNase P and peptidyltransferase • Abzymes- antibodies raised to bind the transition state of a reaction of interest

7. Examples • Ribonuclease P (RNase P) is a type of ribonuclease which cleaves RNA. RNase P is a ribozyme – a ribonucleic acid that acts as a catalyst in the same way that a protein based enzyme would. Its function is to cleave off an extra, or precursor, sequence of RNA on tRNA molecules. • Peptidyltransferase activity is not mediated by any ribosomal proteins but by ribosomal RNA (rRNA), a ribozyme. This RNA relic is the most significant piece of evidence supporting the RNA World hypothesis.

8. History • 1982:Self-splicing in Tetrahymena pre-rRNA (group I intron) • Kruger et al, and Cech, Cell 31, 147-157 (1982) • 1983:RNAse P is a ribozyme • Guerrier-Takada et al, and Altman, Cell, 35, 849-857 (1983)

9. How many ribozyme ? - the hammerhead ribozyme (plant virus) - the hairpin ribozyme (Plant virus) - hepatitis delta ribozyme (human virus) - neurospora VS ribozyme (mitochondrial RNA) - group I and group II intron ribozyme (rRNA and mt RNA) - RNAse P (tRNA maturation) - Ribosome (translation) - Spliceosome ?? (splicing)

10. Ribozymes In vivo self-splicing introns are not catalyst, but true catalyst can be constructed from them, which function in vitro. Ribozymes are much like proteins, and they have a 3-D structure that is required for their function. Ribozymesmeasure many of the same properties as enzymes (kcat, KM, and Vmax). An enzyme that catalyzes a similar reaction to a ribozyme is almost always a better catalysts (kcat/KM). Proteins have more functional groups. Ribozymes are slower at catalyzing the chemistry and have more problems with substrate/product , binding/release. There arealso “selected” DNA catalysts in vitro. They tend to be poorer catalysts than ribozymes (2’ OH’s play an important role in the folding and catalytic properties of ribozymes).

11. Catalytic efficiency, condition - ribozyme follows a Michaelis-Menten kinetics k1 k2 E + S ES E + P k-1 k-1+k2 Km= kcat= 0.5-2 min-1 = 10-5-10-7 M k1 kcat/ Km= 103-106 M-1.min-1 Good catalytic efficiency!! - all ribozyme need cations for activity (Mg2+ ,Mn2+)

12. ABZYMES(Antibody + Enzymes) • Abzymes- antibodies raised to bind the transition state of a reaction of interest

13. ABZYMES(Antibody + Enzymes) Monoclonal antibody  Catalytic activity  Artificial construct  Normal humans  Auto immune diseases PRINCIPLE  Lowering activation energy  Stabilizing less favorable intermediates

14. idea • By exploiting the highly specific antigen binding properties of antibodies, experimental strategies have been devised to produce antibodies that catalyze chemical reactions. These catalytic antibodies, or abzymes, are selected from monoclonal antibodies generated by immunizing mice with haptens that mimic the transition states of enzyme-catalyzed reactions.

15. NATURALLY OCCURING ABZYMES The first natural abzymes were obtained by means of antibody purification from human serum.  Antibodies with protease activity against vasoactif intestinal peptide (VIP)were first isolated in the serum of patients with asthmaasthma..  This VIP-ase activity was also shown to be present on proteins, that are monoclonal human light chains found in urine of patients with multiple myeloma..   DNA hydrolyzing auto antibodies were also isolated from the sera of patients with systemic lupus erythematosusor rheumatoid arthritis..

16. APPLICATIONS • Great potential in the pharmaceutical industries. • Detoxification of cocaine. • Specific targeting of cancer cells unique determinants, called tumor cell antigens, on their surface that are lacking in normal cells. • One application could concern the use of hydrolytic properties of abzymes to activate pro drugs. By targeting this activity in the vicinity to tumor cells, prodrugs could be transformed into cytotoxiccompounds directly on tumor cells. This anti-cancer therapy is designed as Antibody-Directed AbzymeProdrug Therapy (ADAPT). • Inactivate viruses. • Finally, all researches aimed to produce antibodies with a sequence-specific protease activity could open new ways for anti-virus therapy and for the conception of new vaccines.