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MOLECULAR BIOCHEMISTRY II INTRODUCTORY LECTURE

MOLECULAR BIOCHEMISTRY II INTRODUCTORY LECTURE. SYLLABUS AMINO ACID BIOSYNTHESIS ENERGY METABOLISM OBESITY DIABETES ATKINS DIET NUCLEOTIDE METABOLISM DNA STRUCTURE DNA – PROTEIN INTERACTIONS TRANSCRIPTION FACTORS DNA METHYLATION PHOTOSYNTHESIS. SOME CHEMICAL PRINCIPLES TO BE COVERED.

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MOLECULAR BIOCHEMISTRY II INTRODUCTORY LECTURE

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  1. MOLECULAR BIOCHEMISTRY IIINTRODUCTORY LECTURE • SYLLABUS • AMINO ACID BIOSYNTHESIS • ENERGY METABOLISM • OBESITY • DIABETES • 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 C1 UNITS • OXIDATION/REDUCTION REACTIONS

  3. ENZYME CLASSIFICATION • SIX CLASSES ( http://us.expasy.org/enzyme/ ) • NOMENCLATURE COMMITTEE OF INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (1992) • COVALENT CHEMICAL BONDS MADE/BROKEN • OXIDOREDUCTASES • TRANSFERASES • HYDROLASES • LYASES • ISOMERASES • LIGASES • 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 • OXIDOREDUCTASE • TRANSFERASE • HYDROLASE • LYASE • ISOMERASE • LIGASE

  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 + H2O  ADP + Pi + HEAT • Keq = [ADP][Pi] / [ATP] • ∆Ghydrolysis IS RELEASED AS HEAT • HERE THE ENZYME IS ATPase AND IT’S A HYDROLASE

  9. ENERGASE EXAMPLE A SYNTHETASE REACTION: • ATP + GLU + NH3 GLN + ADP + Pi • HERE THE ∆Ghydrolysis IS COUPLED TO ∆Gsynthesis THROUGH A REACTIVE INTERMEDIATE • Keq = [GLN][ADP][Pi] / [ATP][GLU][NH3] = [GLN] / [GLU][NH3] X [ADP][Pi] / [ATP] • AN ENERGASE REACTION: • ATP + STATE 1 + H2O  ADP + STATE 2 + Pi • HERE THE ∆Ghydrolysis IS COUPLED TO ∆Gconformational change • Keq = [STATE 1] / [STATE 2] X [ADP][Pi] / [ATP] • NOTICE SIMILARITY TO Keq 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 • Mullins, J.J. “Six pillars of organic chemistry”, J. Chem. Educ. 2008, 85(1), 83-87

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

  17. AROMATICITY • Mullins, J.J. “Six pillars of organic chemistry”, J. Chem. Educ. 2008, 85(1), 83-87

  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 • N2 IS A VERY STABLE MOLECULE • BOND ENERGY = 941.4 kJ/MOL • COMPARED TO 498.7 kJ/MOL FOR O2 • A SINGLE C=O BOND IN CO2 IS 799 kJ/MOL • HOW IS IT METABOLIZED (“FIXED”)? • THE “NITROGEN CYCLE” • PRODUCTION OF METABOLICALLY USEFUL NITROGEN • NITRITES • NITRATES • AMMONIA

  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: N2 + 8 H+ + 8 e- + 16 ATP + 16 H2O  2 NH3 + H2 + 16 ADP + 16 Pi • REQUIRES ATP AND ELECTRONS • CONTAINS Fe AND Mo

  22. THE NITROGEN CYCLE • ENERGETICALLY COSTLY • NEED 16 ATPs TO “FIX” ONE N2 MOLECULE • COMPARE THIS TO INDUSTRIAL FIXATION: • TEMPERATURE 300o - 500o C • PRESSURE > 300 ATM • METAL CATALYST • NH3 FORMED IS USED IN FORMATION OF • GLUTAMATE (Glu Dehydrogenase) • GLUTAMINE (Gln Synthetase) • EXCESS NH3 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 • NH3 • NO2- • NO3- • SOURCES: • LIGHTNING (10% OF NATURALLY-FIXED N) • FERTILIZERS • DECAY OF ORGANIC MATTER IN SOIL

  24. THE NITROGEN CYCLE • PLANTS, FUNGI, BACTERIA REDUCE NO3-: • A TWO-STEP PROCESS • NO3- + 2H+ + 2e- NO2- + H2O • ENZYME: NITRATE REDUCTASE • NO2- + 8H+ + 6e-  NH4+ + 2H2O • ENZYME: NITRITE REDUCTASE • SOME BACTERIA CAN OXIDIZE NH4+ • “NITRIFICATION” • NH4+ NO2- AND THEN TO NO3- • DENITRIFICATION • CONVERSION OF NO3- TO N2 BY OTHER BACTERIA

  25. THE NITROGEN CYCLE • ATMOSPHERIC N2 IS THE ULTIMATE NITROGEN SOURCE N2 DENITRIFICATION NO3- NITRATE REDUCTASE NITROGENASE NITROGEN FIXATION NO2- NITRITE REDUCTASE NITRIFICATION NH4+

  26. ORGANISMS ASSIMILATE NH3 • ROLE OF GLUTAMINE SYNTHETASE • MICRO-ORGANISMS: ENTRY POINT FOR FIXED N • GLU + ATP + NH4+ GLN + ADP + Pi • IN ALL ORGANISMS, GLN IS AN AMINO GROUP CARRIER • GLUTAMATE SYNTHASE IN BACTERIA, PLANTS • -KETOGLUTARATE + GLN + NADPH + H+ 2 GLU + NADP+ OVERALL RXN’: -KG + NH4+ + ATP + NADPH + H+ GLU + NADP+ + ADP + Pi

  27. THE CENTRAL ROLE OF GLUTAMATE • “GLUTAMATE FAMILY” OF AMINO ACIDS • DEGRADATIVE METABOLISM CONVERGES ON THAT OF GLU • GLU • GLN • PRO • HIS • ARG • ORNITHINE • 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)+ + H2O  -KG + NAD(P)H + NH4+ • 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

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