1. MOLECULAR BIOCHEMISTRY II INTRODUCTORY LECTURE SYLLABUS –AMINO ACID BIOSYNTHESIS –ENERGY METABOLISM OBESITYDIABETES ATKINS DIET –NUCLEOTIDE METABOLISM –DNA STRUCTURE –DNA – PROTEIN INTERACTIONS TRANSCRIPTION FACTORS –DNA METHYLATION –PHOTOSYNTHESIS

2. SOME CHEMICAL PRINCIPLES TO BE COVERED BIOCHEMICAL PATHWAYS –ENZYME CLASSIFICATION –MECHANISMS –REGULATORY CONTROL ROLE OF METAL IONS IN BIOCHEMISTRY PRINCIPLES OF CATALYSIS –TRANSITION STATES COFACTORS –ADDITION OF C 1 UNITS OXIDATION/REDUCTION REACTIONS

3. ENZYME CLASSIFICATION SIX CLASSES ( http://us.expasy.org/enzyme/ ) http://us.expasy.org/enzyme/ –NOMENCLATURE COMMITTEE OF INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (1992) –COVALENT CHEMICAL BONDS MADE/BROKEN OXIDOREDUCTASESTRANSFERASESHYDROLASESLYASESISOMERASESLIGASES ADDITIONAL CLASS (“ENERGASES”) –PHYSICAL REACTIONS –NON-COVALENT PRODUCT-LIKE AND SUBSTRATE-LIKE STATES

4. WHAT CONSTITUTES A CHEMICAL BOND? “…there is a chemical bond between two atoms or groups of atoms in case that the forces acting between them are such as to lead to the formation of an aggregate with sufficient stability to make it convenient for the chemist to consider it as an independent molecular species.” Linus Pauling in “The Nature of the Chemical Bond”

5. SIX TRADITIONAL ENZYME CLASSES CAN YOU RECOGNIZE THE CLASS TO WHICH AN ENZYME BELONGS BY LOOKING AT THE OVERALL REACTION? IN-CLASS EXERCISE –FOR THE FOLLOWING 10 REACTIONS WHICH YOU HAVE ALREADY SEEN THUS FAR IN YOUR STUDY OF BIOCHEMISTRY, INDICATE THE ENZYME BY NAME OR BY CLASS

6. SIX ENZYME CLASSES OXIDOREDUCTASETRANSFERASEHYDROLASELYASEISOMERASELIGASE

7. CATALYSIS OF “PHYSICAL” REACTIONS PRODUCT-LIKE AND SUBSTRATE-LIKE STATES: EXAMPLES : –CHAPERONIN-MEDIATED (PROTEIN FOLDING) –CHROMATIN CONDENSATION –“MOLECULAR MOTOR” OPERATION –DNA PROCESSING BY POLYMERASES –ACTIVE AND CARRIER-MEDIATED TRANSPORT –G-PROTEIN MEDIATED REGULATION OF HORMONE RECEPTORS MEMBRANE TRANSPORTERS (PUMPS) ARE NOW RECOGNIZED AS A SPECIAL CLASS OF ENZYMES “ENERGASES” : TRANSDUCE ENERGY FROM COVALENT BONDS INTO MECHANICAL WORK

8. “ENERGASES” MEDIATE NUCLEOSIDE TRIPHOSPHATE HYDROLYSIS THE FREE ENERGY RELEASED IS COUPLED TO SYSTEM’S CONFORMATIONAL CHANGE ARE ATPases AND GTPases CORRECTLY CLASSIFIED AS “HYDROLASES”? –ATP + H 2 O  ADP + P i + HEAT K eq = [ADP][P i ] / [ATP] ∆G hydrolysis IS RELEASED AS HEAT HERE THE ENZYME IS ATPase AND IT’S A HYDROLASE

9. ENERGASE EXAMPLE A SYNTHETASE REACTION: A SYNTHETASE REACTION: –ATP + GLU + NH 3  GLN + ADP + P i –HERE THE ∆G hydrolysis IS COUPLED TO ∆G synthesis THROUGH A REACTIVE INTERMEDIATE THROUGH A REACTIVE INTERMEDIATE –K eq = [GLN][ADP][P i ] / [ATP][GLU][NH 3 ] = [GLN] / [GLU][NH 3 ] X [ADP][P i ] / [ATP] = [GLN] / [GLU][NH 3 ] X [ADP][P i ] / [ATP] AN ENERGASE REACTION: AN ENERGASE REACTION: –ATP + STATE 1 + H 2 O  ADP + STATE 2 + P i –HERE THE ∆G hydrolysis IS COUPLED TO ∆G conformational change –K eq = [STATE 1] / [STATE 2] X [ADP][P i ] / [ATP] –NOTICE SIMILARITY TO K eq FOR SYNTHETASE REACTION –THERE’S NO CHEMICAL (COVALENT) CHANGE, THOUGH

10. ENZYMES AS MECHANOCHEMICAL PROTEINS THE GIBBS FREE ENERGY OF ATP HYDROLYSIS IS TRANSDUCED INTO A FORM OF USEFUL WORK –TRANSLATION –ROTATION –SOLUTE GRADIENT A RECIPROCAL RELATIONSHIP –ENZYMES USE NON-COVALENT INTERACTIONS TO BREAK COVALENT BONDS –ENERGY FROM BREAKING COVALENT BONDS CAN MODIFY NON-COVALENT INTERACTIONS

11. KEY CONCEPTS IN ORGANIC CHEMISTRY THE “SIX PILLARS” –ELECTRONEGATIVITY –POLAR COVALENT BONDING –STERIC EFFECTS –INDUCTIVE EFFECTS –RESONANCE –AROMATICITY Mullins, J.J. “Six pillars of organic chemistry”, J. Chem. Educ. 2008, 85(1), 83-87

12. ELECTRONEGATIVITY

13. POLAR COVALENT BONDING Mullins, J.J. “Six pillars of organic chemistry”, J. Chem. Educ. 2008, 85(1), 83-87

14. STERIC EFFECTS Mullins, J.J. “Six pillars of organic chemistry”, J. Chem. Educ. 2008, 85(1), 83-87

15. INDUCTIVE EFFECTS Mullins, J.J. “Six pillars of organic chemistry”, J. Chem. Educ. 2008, 85(1), 83-87

16. RESONANCE

17. AROMATICITY

18. SUGGESTION FOR LEARNING BIOCHEMICAL MECHANISMS WHENEVER POSSIBLE, TRY TO RATIONALIZE MECHANISMS USING ONE OR MORE OF THESE “PILLARS”

19. AN INTRODUCTION TO AMINO ACID METABOLISM NITROGEN CYCLE –THE “FIXTATION” OF NITROGEN THE CENTRAL ROLE OF GLUTAMATE

20. THE NITROGEN CYCLE N 2 IS A VERY STABLE MOLECULE –BOND ENERGY = 941.4 kJ/MOL –COMPARED TO 498.7 kJ/MOL FOR O 2 –A SINGLE C=O BOND IN CO 2 IS 799 kJ/MOL HOW IS IT METABOLIZED (“FIXED”)? THE “NITROGEN CYCLE” –PRODUCTION OF METABOLICALLY USEFUL NITROGEN NITRITESNITRATESAMMONIA

21. THE NITROGEN CYCLE N-FIXING ORGANISMS: –ANAEROBES MARINE CYANOBACTERIA “DIAZOTROPHS”DIAZOTROPHS –COLONIZE ROOT NODULES OF LEGUMES –GENUS Rhizobium SYMBIOTIC RELATIONSHIP –ENZYME IS “NITROGENASE” THE NITROGENASE REACTION: N 2 + 8 H + + 8 e - + 16 ATP + 16 H 2 O  2 NH 3 + H 2 + 16 ADP + 16 P i N 2 + 8 H + + 8 e - + 16 ATP + 16 H 2 O  2 NH 3 + H 2 + 16 ADP + 16 P i –REQUIRES ATP AND ELECTRONS –CONTAINS Fe AND Mo

22. THE NITROGEN CYCLE ENERGETICALLY COSTLY –NEED 16 ATPs TO “FIX” ONE N 2 MOLECULE COMPARE THIS TO INDUSTRIAL FIXATION: –TEMPERATURE 300 o - 500 o C –PRESSURE > 300 ATM –METAL CATALYST NH 3 FORMED IS USED IN FORMATION OF –GLUTAMATE(Glu Dehydrogenase) –GLUTAMINE(Gln Synthetase) EXCESS NH 3 EXCRETED INTO SOIL RESTORE USABLE NITROGEN BY PLANTING ALFALFA

23. THE NITROGEN CYCLE MOST PLANTS DO NOT SUPPORT N-FIXING BACTERIA NEED PRE-FIXED NITROGEN SOURCE –NH 3 –NO 2 - –NO 3 - SOURCES: –LIGHTNING (10% OF NATURALLY-FIXED N) –FERTILIZERS –DECAY OF ORGANIC MATTER IN SOIL

24. THE NITROGEN CYCLE PLANTS, FUNGI, BACTERIA REDUCE NO 3 - : –A TWO-STEP PROCESS NO 3 - + 2H + + 2e -  NO 2 - + H 2 O –ENZYME: NITRATE REDUCTASE NO 2 - + 8H + + 6e -  NH 4 + + 2H 2 O –ENZYME: NITRITE REDUCTASE SOME BACTERIA CAN OXIDIZE NH 4 + –“NITRIFICATION” –NH 4 +  NO 2 - AND THEN TO NO 3 - DENITRIFICATION –CONVERSION OF NO 3 - TO N 2 BY OTHER BACTERIA

25. THE NITROGEN CYCLE ATMOSPHERIC N 2 IS THE ULTIMATE NITROGEN SOURCE N 2 NH 4 + NO 3 - NO 2 - NITROGEN FIXATION NITRIFICATION DENITRIFICATION NITROGENASE NITRATE REDUCTASE NITRITE REDUCTASE

26. ORGANISMS ASSIMILATE NH 3 ROLE OF GLUTAMINE SYNTHETASE –MICRO-ORGANISMS: ENTRY POINT FOR FIXED N –GLU + ATP + NH 4 +  GLN + ADP + P i IN ALL ORGANISMS, GLN IS AN AMINO GROUP CARRIER GLUTAMATE SYNTHASE IN BACTERIA, PLANTS –  -KETOGLUTARATE + GLN + NADPH + H +  2 GLU + NADP + OVERALL RXN’:  -KG + NH 4 + + ATP + NADPH + H +  GLU + NADP + + ADP + P i

27. THE CENTRAL ROLE OF GLUTAMATE “GLUTAMATE FAMILY” OF AMINO ACIDS –DEGRADATIVE METABOLISM CONVERGES ON THAT OF GLU GLUGLNPROHISARGORNITHINE GLU IS THE PRECURSOR OF –PRO –ORNITHINE –ARG GLU/  -KG ARE TRANSAMINATION PARTNERS –AMINO ACID +  -KG  GLU +  -KETOACID OXIDATIVE DEAMINATION OF GLU (GLU DEHYDROGENASE) GLU + NAD(P) + + H 2 O   -KG + NAD(P)H + NH 4 + N-ACETYLGLUTAMATE SYNTHESIS –ALLOSTERICALLY REGULATES CPS I OF UREA CYCLE –GLU + ACETYL-CoA  N-ACETYL GLUTAMATE

28. Kelly A., Stanley CA. (2001). “Disorders of Glutamate Metabolism”. Mental Retard- Ation and Developmental Disorders. 7:287-295.

29. CLOSING POINTS HIGH ENERGY COSTS TO FIX NITROGEN –ITS USE MUST BE CAREFULLY CONTROLLED GLU AND GLN ARE PIVOTAL IN AMINO GROUP TRANSFER –GLU OFTEN DONATES THE AMINO GROUP –GLN STORES, CARRIES AMINO GROUPS TRANSAMINASES –CATALYSTS FOR TRANSFER OF AMINO GROUPS TO α- KETOACIDS –FREELY REVERSIBLE REACTIONS  IMPORTANT IN BOTH SYNTHETIC AND DEGRADATIVE PATHWAYS