MOLECULAR BIOCHEMISTRY IIMOLECULAR BIOCHEMISTRY IIINTRODUCTORY LECTUREINTRODUCTORY LECTURE

SYLLABUSSYLLABUS– AMINO ACID BIOSYNTHESISAMINO ACID BIOSYNTHESIS– ENERGY METABOLISMENERGY METABOLISM

OBESITYOBESITYDIABETESDIABETESATKINS DIETATKINS DIET

– NUCLEOTIDE METABOLISMNUCLEOTIDE METABOLISM– DNA STRUCTUREDNA STRUCTURE– DNA – PROTEIN INTERACTIONSDNA – PROTEIN INTERACTIONS

TRANSCRIPTION FACTORSTRANSCRIPTION FACTORS– DNA METHYLATIONDNA METHYLATION– PHOTOSYNTHESISPHOTOSYNTHESIS

SOME CHEMICAL PRINCIPLES TO BE SOME CHEMICAL PRINCIPLES TO BE COVEREDCOVERED

BIOCHEMICAL PATHWAYSBIOCHEMICAL PATHWAYS– ENZYME CLASSIFICATIONENZYME CLASSIFICATION– MECHANISMSMECHANISMS– REGULATORY CONTROLREGULATORY CONTROL

ROLE OF METAL IONS IN BIOCHEMISTRYROLE OF METAL IONS IN BIOCHEMISTRYPRINCIPLES OF CATALYSISPRINCIPLES OF CATALYSIS– TRANSITION STATESTRANSITION STATES

COFACTORSCOFACTORS– ADDITION OF CADDITION OF C11 UNITS UNITS

OXIDATION/REDUCTION REACTIONSOXIDATION/REDUCTION REACTIONS

ENZYME CLASSIFICATIONENZYME CLASSIFICATION

SIX CLASSES ( SIX CLASSES ( http://us.expasy.org/enzyme/http://us.expasy.org/enzyme/ ) )– NOMENCLATURE COMMITTEE OF INTERNATIONAL UNION NOMENCLATURE COMMITTEE OF INTERNATIONAL UNION

OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (1992)OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (1992)– COVALENT CHEMICAL BONDS MADE/BROKENCOVALENT CHEMICAL BONDS MADE/BROKEN

OXIDOREDUCTASESOXIDOREDUCTASESTRANSFERASESTRANSFERASESHYDROLASESHYDROLASESLYASESLYASESISOMERASESISOMERASESLIGASESLIGASES

ADDITIONAL CLASS (“ENERGASES”)ADDITIONAL CLASS (“ENERGASES”)– PHYSICAL REACTIONSPHYSICAL REACTIONS– NON-COVALENT PRODUCT-LIKE AND SUBSTRATE-LIKE NON-COVALENT PRODUCT-LIKE AND SUBSTRATE-LIKE

STATESSTATES

WHAT CONSTITUTES A CHEMICAL WHAT CONSTITUTES A CHEMICAL BOND?BOND?

“…“…there is a chemical bond between two atoms or there is a chemical bond between two atoms or groups of atoms in case that the forces acting between groups of atoms in case that the forces acting between them are such as to lead to the formation of an them are such as to lead to the formation of an aggregate with sufficient stability to make it convenient aggregate with sufficient stability to make it convenient for the chemist to consider it as an independent for the chemist to consider it as an independent molecular species.”molecular species.”

Linus Pauling in “The Nature of the Chemical Bond”Linus Pauling in “The Nature of the Chemical Bond”

SIX TRADITIONAL ENZYME CLASSESSIX TRADITIONAL ENZYME CLASSES

CAN YOU RECOGNIZE THE CLASS TO CAN YOU RECOGNIZE THE CLASS TO WHICH AN ENZYME BELONGS BY WHICH AN ENZYME BELONGS BY LOOKING AT THE OVERALL LOOKING AT THE OVERALL REACTION?REACTION?IN-CLASS EXERCISEIN-CLASS EXERCISE– FOR THE FOLLOWING 10 REACTIONS FOR THE FOLLOWING 10 REACTIONS

WHICH YOU HAVE ALREADY SEEN THUS WHICH YOU HAVE ALREADY SEEN THUS FAR IN YOUR STUDY OF BIOCHEMISTRY, FAR IN YOUR STUDY OF BIOCHEMISTRY, INDICATE THE ENZYME BY NAME OR BY INDICATE THE ENZYME BY NAME OR BY CLASSCLASS

SIX ENZYME CLASSESSIX ENZYME CLASSES

OXIDOREDUCTASEOXIDOREDUCTASE

TRANSFERASETRANSFERASE

HYDROLASEHYDROLASE

LYASELYASE

ISOMERASEISOMERASE

LIGASELIGASE

CATALYSIS OF “PHYSICAL” REACTIONSCATALYSIS OF “PHYSICAL” REACTIONS

PRODUCT-LIKE AND SUBSTRATE-LIKE STATES: PRODUCT-LIKE AND SUBSTRATE-LIKE STATES: EXAMPLES :EXAMPLES :– CHAPERONIN-MEDIATED (PROTEIN FOLDING)CHAPERONIN-MEDIATED (PROTEIN FOLDING)– CHROMATIN CONDENSATIONCHROMATIN CONDENSATION– ““MOLECULAR MOTOR” OPERATIONMOLECULAR MOTOR” OPERATION– DNA PROCESSING BY POLYMERASESDNA PROCESSING BY POLYMERASES– ACTIVE AND CARRIER-MEDIATED TRANSPORTACTIVE AND CARRIER-MEDIATED TRANSPORT– G-PROTEIN MEDIATED REGULATION OF HORMONE G-PROTEIN MEDIATED REGULATION OF HORMONE

RECEPTORSRECEPTORS

MEMBRANE TRANSPORTERS (PUMPS) ARE NOW MEMBRANE TRANSPORTERS (PUMPS) ARE NOW RECOGNIZED AS A SPECIAL CLASS OF ENZYMESRECOGNIZED AS A SPECIAL CLASS OF ENZYMES““ENERGASES” : TRANSDUCE ENERGY FROM ENERGASES” : TRANSDUCE ENERGY FROM COVALENT BONDS INTO MECHANICAL WORKCOVALENT BONDS INTO MECHANICAL WORK

““ENERGASES”ENERGASES”

MEDIATE NUCLEOSIDE TRIPHOSPHATE MEDIATE NUCLEOSIDE TRIPHOSPHATE HYDROLYSISHYDROLYSIS

THE FREE ENERGY RELEASED IS COUPLED THE FREE ENERGY RELEASED IS COUPLED TO SYSTEM’S CONFORMATIONAL CHANGE TO SYSTEM’S CONFORMATIONAL CHANGE

ARE ATPases AND GTPases CORRECTLY ARE ATPases AND GTPases CORRECTLY CLASSIFIED AS “HYDROLASES”?CLASSIFIED AS “HYDROLASES”?– ATP + HATP + H22O O ADP + P ADP + Pii + HEAT + HEAT

KKeqeq = [ADP][P = [ADP][Pii] / [ATP]] / [ATP]

∆∆GGhydrolysis hydrolysis IS RELEASED AS HEAT IS RELEASED AS HEAT

HERE THE ENZYME IS ATPase AND IT’S A HYDROLASEHERE THE ENZYME IS ATPase AND IT’S A HYDROLASE

ENERGASE EXAMPLEENERGASE EXAMPLE

A SYNTHETASE REACTION:A SYNTHETASE REACTION:– ATP + GLU + NHATP + GLU + NH33 GLN + ADP + P GLN + ADP + Pii – HERE THE ∆GHERE THE ∆Ghydrolysishydrolysis IS COUPLED TO ∆G IS COUPLED TO ∆Gsynthesissynthesis

THROUGH A REACTIVE INTERMEDIATETHROUGH A REACTIVE INTERMEDIATE– KKeqeq = [GLN][ADP][P = [GLN][ADP][Pii] / [ATP][GLU][NH] / [ATP][GLU][NH33]]

= [GLN] / [GLU][NH= [GLN] / [GLU][NH33] X [ADP][P] X [ADP][Pii] / [ATP]] / [ATP]

AN ENERGASE REACTION:AN ENERGASE REACTION:– ATP + STATE 1 + HATP + STATE 1 + H22O O ADP + STATE 2 + P ADP + STATE 2 + Pii – HERE THE ∆GHERE THE ∆Ghydrolysishydrolysis IS COUPLED TO ∆G IS COUPLED TO ∆Gconformational changeconformational change

– KKeqeq = [STATE 1] / [STATE 2] X [ADP][P = [STATE 1] / [STATE 2] X [ADP][P ii] / [ATP]] / [ATP]– NOTICE SIMILARITY TO KNOTICE SIMILARITY TO Keqeq FOR SYNTHETASE REACTION FOR SYNTHETASE REACTION– THERE’S NO CHEMICAL (COVALENT) CHANGE, THOUGHTHERE’S NO CHEMICAL (COVALENT) CHANGE, THOUGH

ENZYMES AS MECHANOCHEMICAL ENZYMES AS MECHANOCHEMICAL PROTEINSPROTEINS

THE GIBBS FREE ENERGY OF ATP THE GIBBS FREE ENERGY OF ATP HYDROLYSIS IS HYDROLYSIS IS TRANSDUCEDTRANSDUCED INTO A FORM INTO A FORM OF USEFUL WORKOF USEFUL WORK– TRANSLATIONTRANSLATION– ROTATIONROTATION– SOLUTE GRADIENTSOLUTE GRADIENT

A RECIPROCAL RELATIONSHIPA RECIPROCAL RELATIONSHIP– ENZYMES USE NON-COVALENT INTERACTIONS TO BREAK ENZYMES USE NON-COVALENT INTERACTIONS TO BREAK

COVALENT BONDSCOVALENT BONDS– ENERGY FROM BREAKING COVALENT BONDS CAN ENERGY FROM BREAKING COVALENT BONDS CAN

MODIFY NON-COVALENT INTERACTIONSMODIFY NON-COVALENT INTERACTIONS

KEY CONCEPTS IN ORGANIC CHEMISTRYKEY CONCEPTS IN ORGANIC CHEMISTRY

THE “SIX PILLARS”THE “SIX PILLARS”– ELECTRONEGATIVITYELECTRONEGATIVITY– POLAR COVALENT BONDINGPOLAR COVALENT BONDING– STERIC EFFECTSSTERIC EFFECTS– INDUCTIVE EFFECTSINDUCTIVE EFFECTS– RESONANCERESONANCE– AROMATICITYAROMATICITY

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

ELECTRONEGATIVITYELECTRONEGATIVITY

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

POLAR COVALENT BONDINGPOLAR COVALENT BONDING

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

STERIC EFFECTSSTERIC EFFECTS

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

INDUCTIVE EFFECTSINDUCTIVE EFFECTS

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

RESONANCERESONANCE

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

AROMATICITYAROMATICITY

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

SUGGESTION FOR LEARNING BIOCHEMICAL SUGGESTION FOR LEARNING BIOCHEMICAL MECHANISMSMECHANISMS

WHENEVER POSSIBLE, TRY TO RATIONALIZE WHENEVER POSSIBLE, TRY TO RATIONALIZE MECHANISMS USING ONE OR MORE OF THESE MECHANISMS USING ONE OR MORE OF THESE “PILLARS”“PILLARS”

AN INTRODUCTION TO AMINO AN INTRODUCTION TO AMINO ACID METABOLISMACID METABOLISM

NITROGEN CYCLENITROGEN CYCLE– THE “FIXTATION” OF NITROGENTHE “FIXTATION” OF NITROGEN

THE CENTRAL ROLE OF GLUTAMATETHE CENTRAL ROLE OF GLUTAMATE

THE NITROGEN CYCLETHE NITROGEN CYCLE

NN22 IS A VERY STABLE MOLECULE IS A VERY STABLE MOLECULE– BOND ENERGY = 941.4 kJ/MOLBOND ENERGY = 941.4 kJ/MOL– COMPARED TO 498.7 kJ/MOL FOR OCOMPARED TO 498.7 kJ/MOL FOR O22 – A SINGLE C=O BOND IN COA SINGLE C=O BOND IN CO22 IS 799 kJ/MOL IS 799 kJ/MOL

HOW IS IT METABOLIZED (“FIXED”)?HOW IS IT METABOLIZED (“FIXED”)?THE “NITROGEN CYCLE”THE “NITROGEN CYCLE”– PRODUCTION OF METABOLICALLY USEFUL PRODUCTION OF METABOLICALLY USEFUL

NITROGENNITROGENNITRITESNITRITESNITRATESNITRATESAMMONIAAMMONIA

THE NITROGEN CYCLETHE NITROGEN CYCLE

N-FIXING ORGANISMS:N-FIXING ORGANISMS:– ANAEROBESANAEROBES

MARINE CYANOBACTERIAMARINE CYANOBACTERIA““DIAZOTROPHS” DIAZOTROPHS”

DIAZOTROPHSDIAZOTROPHS– COLONIZE ROOT NODULES OF LEGUMESCOLONIZE ROOT NODULES OF LEGUMES– GENUS GENUS RhizobiumRhizobium

SYMBIOTIC RELATIONSHIPSYMBIOTIC RELATIONSHIP– ENZYME IS “NITROGENASE”ENZYME IS “NITROGENASE”

THE NITROGENASE REACTION:THE NITROGENASE REACTION: NN22 + 8 H + 8 H++ + 8 e + 8 e-- + 16 ATP + 16 H + 16 ATP + 16 H22O O 2 NH 2 NH33 + H + H22 + 16 ADP + 16 P + 16 ADP + 16 Pii

– REQUIRES ATP AND ELECTRONSREQUIRES ATP AND ELECTRONS– CONTAINS Fe AND MoCONTAINS Fe AND Mo

THE NITROGEN CYCLETHE NITROGEN CYCLE

ENERGETICALLY COSTLYENERGETICALLY COSTLY– NEED 16 ATPs TO “FIX” ONE NNEED 16 ATPs TO “FIX” ONE N22 MOLECULE MOLECULE

COMPARE THIS TO INDUSTRIAL FIXATION:COMPARE THIS TO INDUSTRIAL FIXATION:– TEMPERATURE 300TEMPERATURE 300oo

- 500 - 500oo C C– PRESSURE > 300 ATMPRESSURE > 300 ATM– METAL CATALYSTMETAL CATALYST

NHNH33 FORMED IS USED IN FORMATION OF FORMED IS USED IN FORMATION OF– GLUTAMATEGLUTAMATE (Glu Dehydrogenase)(Glu Dehydrogenase)– GLUTAMINEGLUTAMINE (Gln Synthetase)(Gln Synthetase)

EXCESS NHEXCESS NH33 EXCRETED INTO SOIL EXCRETED INTO SOILRESTORE USABLE NITROGEN BY PLANTING RESTORE USABLE NITROGEN BY PLANTING ALFALFAALFALFA

THE NITROGEN CYCLETHE NITROGEN CYCLE

MOST PLANTS DO NOT SUPPORT N-FIXING MOST PLANTS DO NOT SUPPORT N-FIXING BACTERIABACTERIANEED PRE-FIXED NITROGEN SOURCENEED PRE-FIXED NITROGEN SOURCE– NHNH33

– NONO22--

– NONO33--

SOURCES:SOURCES:– LIGHTNING (10% OF NATURALLY-FIXED N)LIGHTNING (10% OF NATURALLY-FIXED N)– FERTILIZERSFERTILIZERS– DECAY OF ORGANIC MATTER IN SOILDECAY OF ORGANIC MATTER IN SOIL

THE NITROGEN CYCLETHE NITROGEN CYCLE

PLANTS, FUNGI, BACTERIA REDUCE NOPLANTS, FUNGI, BACTERIA REDUCE NO33--::

– A TWO-STEP PROCESSA TWO-STEP PROCESS

NONO33-- + 2H + 2H++ + 2e + 2e-- NO NO22

-- + H + H22OO– ENZYME: NITRATE REDUCTASEENZYME: NITRATE REDUCTASE

NONO22- - + 8H + 8H++ + 6e + 6e-- NH NH44

++ + 2H + 2H22OO

– ENZYME: NITRITE REDUCTASEENZYME: NITRITE REDUCTASE

SOME BACTERIA CAN OXIDIZE NHSOME BACTERIA CAN OXIDIZE NH44++

– ““NITRIFICATION”NITRIFICATION”– NHNH44

++ NO NO22-- AND THEN TO NO AND THEN TO NO33

--

DENITRIFICATIONDENITRIFICATION– CONVERSION OF NOCONVERSION OF NO33

-- TO N TO N22 BY OTHER BACTERIA BY OTHER BACTERIA

THE NITROGEN CYCLETHE NITROGEN CYCLE

ATMOSPHERIC NATMOSPHERIC N22 IS THE ULTIMATE NITROGEN SOURCE IS THE ULTIMATE NITROGEN SOURCE

N2

NH4+

NO3-

NO2-

NITROGEN FIXATION

NITRIFICATION

DENITRIFICATION

NITROGENASENITRATEREDUCTASE

NITRITEREDUCTASE

ORGANISMS ASSIMILATE NHORGANISMS ASSIMILATE NH33

ROLE OF GLUTAMINE SYNTHETASEROLE OF GLUTAMINE SYNTHETASE

– MICRO-ORGANISMS: ENTRY POINT FOR FIXED NMICRO-ORGANISMS: ENTRY POINT FOR FIXED N

– GLU + ATP + NHGLU + ATP + NH44++ GLN + ADP + P GLN + ADP + P ii

IN ALL ORGANISMS, GLN IS AN AMINO GROUP IN ALL ORGANISMS, GLN IS AN AMINO GROUP CARRIERCARRIER

GLUTAMATE SYNTHASE IN BACTERIA, PLANTSGLUTAMATE SYNTHASE IN BACTERIA, PLANTS -KETOGLUTARATE + GLN + NADPH + H-KETOGLUTARATE + GLN + NADPH + H++ 2 GLU + 2 GLU +

NADP NADP++

OVERALL RXN’: OVERALL RXN’:

-KG + NH-KG + NH44++ + ATP + NADPH + H + ATP + NADPH + H++ GLU + NADP GLU + NADP++ + +

ADP + PADP + Pii

THE CENTRAL ROLE OF GLUTAMATETHE CENTRAL ROLE OF GLUTAMATE

““GLUTAMATE FAMILY” OF AMINO ACIDSGLUTAMATE FAMILY” OF AMINO ACIDS– DEGRADATIVE METABOLISM CONVERGES ON THAT OF GLUDEGRADATIVE METABOLISM CONVERGES ON THAT OF GLU

GLUGLUGLNGLNPROPROHISHISARGARGORNITHINEORNITHINE

GLU IS THE PRECURSOR OFGLU IS THE PRECURSOR OF– PROPRO– ORNITHINEORNITHINE– ARGARG

GLU/GLU/-KG ARE TRANSAMINATION PARTNERS-KG ARE TRANSAMINATION PARTNERS– AMINO ACID + AMINO ACID + -KG -KG GLU + GLU + -KETOACID-KETOACID

OXIDATIVE DEAMINATION OF GLU (GLU DEHYDROGENASE)OXIDATIVE DEAMINATION OF GLU (GLU DEHYDROGENASE)GLU + NAD(P)GLU + NAD(P)++ + H + H22O O -KG + NAD(P)H + NH-KG + NAD(P)H + NH44

++

N-ACETYLGLUTAMATE SYNTHESISN-ACETYLGLUTAMATE SYNTHESIS– ALLOSTERICALLY REGULATES CPS I OF UREA CYCLEALLOSTERICALLY REGULATES CPS I OF UREA CYCLE– GLU + ACETYL-CoA GLU + ACETYL-CoA N-ACETYL GLUTAMATE N-ACETYL GLUTAMATE

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

CLOSING POINTSCLOSING POINTS

HIGH ENERGY COSTS TO FIX NITROGENHIGH ENERGY COSTS TO FIX NITROGEN– ITS USE MUST BE CAREFULLY CONTROLLEDITS USE MUST BE CAREFULLY CONTROLLED

GLU AND GLN ARE PIVOTAL IN AMINO GROUP GLU AND GLN ARE PIVOTAL IN AMINO GROUP TRANSFERTRANSFER– GLU OFTEN DONATES THE AMINO GROUPGLU OFTEN DONATES THE AMINO GROUP– GLN STORES, CARRIES AMINO GROUPSGLN STORES, CARRIES AMINO GROUPS

TRANSAMINASESTRANSAMINASES– CATALYSTS FOR TRANSFER OF AMINO GROUPS TO CATALYSTS FOR TRANSFER OF AMINO GROUPS TO αα--

KETOACIDSKETOACIDS– FREELY REVERSIBLE REACTIONSFREELY REVERSIBLE REACTIONS

IMPORTANT IN BOTH SYNTHETIC AND DEGRADATIVE IMPORTANT IN BOTH SYNTHETIC AND DEGRADATIVE PATHWAYSPATHWAYS