AMINO ACID BIOSYNTHESIS

1. AMINO ACID BIOSYNTHESIS NON-ESSENTIAL AMINO ACIDS ESSENTIAL AMINO ACIDS SINGLE CARBON TRANSFERS WITH THF PHYSIOLOGIC AMINES

2. AMINO ACID BIOSYNTHESIS • “FIXING” OF ATMOSPHERIC N2 • DIAZOTROPHS FIX N2 TO NH3 • IN MICRO-ORGANISMS, PLANTS, LOWER ANIMALS: • GLU DEHYDROGENASE RXN • GLU + NAD(P)+ + H2O  -KG + NH3 + NAD(P)H + H+ • REVERSE RXN  GLU • GLU SYNTHASE RXN’  GLU • NADPH + H+ + GLN + -KG  2 GLU + NADP+

3. AMINO ACID BIOSYNTHESIS • DOES THE GLU DEHYDROGENASE RXN’ WORK IN REVERSE IN MAMMALS? • THERE IS SOME CONTROVERSY ABOUT THIS • THE HYPERAMMONEMIA/HYPERINSULINEMIA SYNDROME (HI/HA) IS CAUSED BY A MUTATION IN GDH THAT  A GAIN IN FUNCTION • SUGGESTS THAT THE PREFERRED DIRECTION IS TOWARD THE RIGHT • DEPENDING UPON THE ORGANISM, THE GLU DEHYDROGENASE MIGHT BE CLOSE TO EQUILIBRIUM, OR FAVORED TO THE RIGHT OR LEFT • SO, PREFORMED -AMINO NITROGEN, IN THE FORM OF GLU, MUST BE CONSIDERED AN ESSENTIAL NUTRIENT

4. AMINO ACID BIOSYNTHESIS • ESSENTIAL AMINO ACIDS *ARGININE METHIONINE HISTIDINE PHENYLALANINE ISOLEUCINE THREONINE LEUCINE TRYPTOPHAN LYSINE VALINE • NOTE • ARG IS ESSENTIAL IN INFANTS AND CHILDREN • MOST SYNTHESIZED ARG  ORNITHINE AND UREA VIA THE UREA CYCLE

5. AMINO ACID BIOSYNTHESIS • NONESSENTIAL AMINO ACIDS ALANINE GLUTAMINE ASPARAGINE GLYCINE ASPARTATE PROLINE *CYSTEINE SERINE GLUTAMATE *TYROSINE • NOTE: • CYS GETS ITS SULFUR ATOM FROM MET • TYR IS HYDROXYLATED PHE • SO IT’S NOT REALLY NONESSENTIAL

6. AMINO ACID BIOSYNTHESIS • ALL ARE SYNTHESIZED FROM COMMON METABOLIC INTERMEDIATES • NON-ESSENTIAL • TRANSAMINATION OF -KETOACIDS THAT ARE AVAILABLE AS COMMON INTERMEDIATES • ESSENTIAL • THEIR -KETOACIDS ARE NOT COMMON INTERMEDIATES (ENZYMES NEEDED TO FORM THEM ARE LACKING) • SO TRANSAMINATION ISN’T AN OPTION • BUT THEY ARE PRESENT IN COMMON PATHWAYS OF MICRO-ORGANISMS AND PLANTS

7. AMINO ACID BIOSYNTHESIS OVERVIEW(USE OF COMMON INTERMEDIATES) GLUCOSE  GLUC-6-PHOSPHATE    RIB-5-PHOS→ HIS   3-PHOSPHOGLYCERATE  SERINE    GLYCINE E-4-PHOS + PEP CYSTEINE   PHE→TYR PYRUVATE ALA TRP VAL CITRATE LEU, ILE ↓ OXALOACETATE, -KETOGLUTARATE ASP, ASN, GLU, GLN, PRO, ARG,LYS, THR, MET

8. SYNTHESIS OF NON-ESSENTIAL AMINO ACIDS • ALL (EXCEPT TYR) SYNTHESIZED FROM COMMON INTERMEDIATES SYNTHESIZED IN CELL • PYRUVATE • OXALOACETATE • -KETOGLUTARATE • 3-PHOSPHOGLYCERATE

9. SYNTHESIS OF NON-ESSENTIAL AMINO ACIDS • TRANSAMINATION REACTIONS: ONE STEP • PYRUVATE + AA  ALANINE + -KETOACID • OXALOACETATE + AA  ASPARTATE + -KETOACID • -KETOGLUTARATE + AA  GLUTAMATE + -KETOACID • TRANSAMINASES: EQUILIBRATE AMINO GROUPS REQUIRE PYRIDOXAL PHOSPHATE (PLP) • ALL AAs, EXCEPT LYS, CAN BE TRANSAMINATED • MOST TRANSAMINASES GENERATE GLU OR ASP • WHY? • LOOK AT MECHANISM OF PLP (PAGE 987 IN TEXT)

10. A C B

12. SYNTHESIS OF NONESSENTIAL AMINO ACIDS • ATP-DEPENDENT AMIDATION OF ASP, GLU •  ASN, GLN • GLU + ATP + NH3  GLN + ADP + Pi • GLUTAMINE SYNTHETASE • NH3 IS TOXIC; IT’S STORED AS GLN • GLN DONATES AMINO GPS IN MANY REACTIONS • ASP + ATP + GLN  ASN + AMP + PPi + GLU • ASPARAGINE SYNTHETASE

13. SYNTHESIS OF NONESSENTIAL AMINO ACIDS • NITROGEN METABOLISM IS CONTROLLED BY REGULATION OF GLUTAMINE SYNTHETASE • IN MAMMALS, GLN SYNTHETASES ACTIVATED BY -KG • EXCESS AAs TRANSAMINATED TO GLU • OXIDATIVE DEAMINATION OF GLU  -KG + NH3 • NH3 UREA OR GLN (STORAGE) • -KG IS A SIGNAL THAT ACTIVATES GLN SYNTHETASE

14. BACTERIAL GLUTAMINE SYNTHETASE • VERY DETAILED CONTROL SYSTEM • 12 IDENTICAL SUBUNITS (HEX PRISM) • ALLOSTERIC CONTROL • 9 FEEDBACK INHIBITORS (CUMULATIVE INH) • INDIVIDUAL BINDING SITES • 6 ARE END-PRODS OF PATHWAYS FROM GLN • HIS, TRP, CARBAMOYL PHOSPHATE, AMP, CTP, GLUCOSAMINE-6-PHOSPHATE • 3 REFLECT CELL’S N LEVEL (ALA, SER, GLY) • ALSO COVALENTLY MODIFIED BY ADENYLYLATION

15. BACTERIAL GLUTAMINE SYNTHETASE • BRIEF REVIEW: REGULATING ENZYME ACTIVITY • NEAR-EQUILIBRIUM (REVERSIBLE) • REACTANTS, PRODUCTS ~ EQUIL. VALUES • ENZYMES ACT QUICKLY TO RESTORE EQUIL. • RATES REGULATED BY [REACT], [PROD] • FAR FROM EQUILIBRIUM (IRREVERSIBLE) • ENZYME SATURATED • NOT ENOUGH ACTIVITY TO ALLOW EQUIL. • RATE INSENSITIVE TO [REACT], [PROD] •  “STEADY STATE” (CONSTANT FLUX) • “RATE-DETERMINING STEP”

16. BACTERIAL GLUTAMINE SYNTHETASE • BRIEF REVIEW: REGULATING ENZYME ACTIVITY CONTROL OF ENZYME ACTIVITY • ALLOSTERIC REGULATION • COVALENT MODIFICATION • GENETIC CONTROL • AT LEVEL OF TRANSCRIPTION

17. BACTERIAL GLUTAMINE SYNTHETASE • SEE REGULATORY DIAGRAM (PAGE 1035) • ADENYLYLATION OF A SPECIFIC TYR RESIDUE •  LESS ACTIVITY OF THE ENZYME • ENZYME IS ADENYLYLTRANSFERASE IN A COMPLEX WITH A TETRAMERIC REGULATORY PROTEIN, PII • URIDYLYLATION OF PII (AT A TYR)  DEADENYLYLATION • A URIDYL-REMOVING ENZYME RESULTS IN ADENYLYLTRANSFERASE CATALYZING ADENYLYLATION OF GLN SYNTHETASE

18. BACTERIAL GLUTAMINE SYNTHETASE • SEE REGULATORY DIAGRAM (PAGE 1035) • WHAT CONTROLS ACTIVITY OF URIDYLYL TRANSFERASE? • ACTIVATED BY -KG AND ATP • DEACTIVATED BY GLN AND Pi • URIDYL-REMOVING ENZYME INSENSITIVE TO THESE

20. BACTERIAL GLUTAMINE SYNTHETASE • IN-CLASS EXERCISE EXPLAIN THE SIGNIFICANCE OF -KG AS AN ACTIVATOR OF GLUTAMINE SYNTHETASE SHOW, IN DETAIL, THE EFFECT OF  LEVEL OF -KG ON THIS ENZYME. DO THE SAME FOR ATP, GLN AND Pi

21. NONESSENTIAL AMINO ACID SYNTHESIS • PRO, ORNITHINE, ARG ARE DERIVED FROM GLUTAMATE • NOTE: 7 OF THE 10 “NONESSENTIALS” ARE ULTIMATELY DERIVED FROM PYR, -KG AND OXALOACETATE • SEE PATHWAYS ON PAGE 1036 • HIGHLIGHTS: • STEP 1: ACTIVATE GLU; A KINASE • GLUTAMATE-5-SEMIALDEHYDE BRANCH POINT • SPONTANEOUS CYCLIZATION TO AN INTERNAL SCHIFF BASE  PRO • TRANSAMINATION TO ORNITHINE  ARG IN UREA CYCLE • SCHIFF BASE: AMINE + (ALDEHYDE OR KETONE)  IMINE (CONTAINS A C=N BOND)

22. NONESSENTIAL AMINO ACID SYNTHESIS • 3-PHOSPHOGLYCERATE IS PRECURSOR OF • SER (A 3-STEP PATHWAY) (1) 3-PG + NAD+ 3-PHOSPHOHYDROXYPYRUVATE + NADH + H+ (2) 3-PHP + GLU  3-PHOSPHOSERINE + -KG (3) 3-PHOSPHOSERINE + H2O  SER + Pi • GLY (2 DIFFERENT WAYS) (1) SER + THF  GLY + N5,N10 – METHYLENE-THF (DIRECT) (2) N5,N10 – METHYLENE-THF + CO2 + NH4+  GLY + THF (CONDENSATION)

24. NONESSENTIAL AMINO ACID SYNTHESIS • CYSTEINE • SER + HOMOCYSTEINE  CYSTATHIONINE • HOMOCYSTEINE IS A BREAKDOWN PRODUCT OF METHIONINE • CYSTATHIONINE  -KETOBUTYRATE + CYS • NOTE: -SH GROUP COMES FROM MET • SO CYS IS ACTUALLY AN ESSENTIAL AMINO ACID

25. NONESSENTIAL AMINO ACID SYNTHESIS • SUMMARY POINT: • ALL NONESSENTIALS (EXCEPT TYR) ARE DERIVED FROM ONE OF THE FOLLOWING COMMON INTERMEDIATES: • PYRUVATE • OXALOACETATE • -KG • 3-PHOSPHOGLYCERATE

26. IN-CLASS EXERCISE • WHICH OF THE 4 AMINO ACID INTERMEDIATES OF THE UREA CYCLE IS ESSENTIAL IN CHILDREN? • OUTLINE A PATHWAY BY WHICH ADULTS CAN SYNTHESIZE THIS AA FROM 1 GLUCOSE MOLECULE. • HINTS: YOU WILL NEED TO CONSIDER THE FOLLOWING METABOLIC PATHWAYS: • GLYCOLYTIC • GLUCONEOGENIC • CITRIC ACID CYCLE • GLUTAMATE DEHYDROGENASE REACTION • ASSUME IT CAN GO IN REVERSE DIRECTION • ORNITHINE PRODUCTION • UREA CYCLE

27. TRANSFER OF C1 UNITS TO METABOLIC PRECURSORS • MOST CARBOXYLATION REACTIONS USE A BIOTIN COFACTOR • EXAMPLE: PYRUVATE CARBOXYLASE REACTION • S-ADENOSYLMETHIONINE (SAM) AS A METHYLATING AGENT • CYTOSINE METHYLATION OF CpGs IN GENE PROMOTER REGIONS • TETRAHYDROFOLATES • CAN TRANSFER SINGLE C UNITS IN A NUMBER OF DIFFERENT OXIDATION STATES

28. TETRAHYDROFOLATES • REVIEW STRUCTURE (PAGE 1028 OF TEXT) • FOCUS ON HETEROCYCLIC RING STRUCTURE • 2-AMINO-4-OXO-6-METHYLPTERIN • NOTICE THE NUMBERING OF THE ATOMS • LOOK AT N5 • PABA JOINS TO 2-AMINO-4-OXO-6-METHYLPTERIN TO FORM PTEROIC ACID • FIND N10 • COVALENT ATTACHMENT OF C1 UNITS AT • N5 • N10 • BOTH

29. TETRAHYDROFOLATE • THREE DIFFERENT OXIDATION STATES • METHANOL AT N5 • METHYL (-CH3) • FORMALDEHYDE AT N5,N10 • METHYLENE (-CH2-) • FORMATE • FORMYL (-CH=O) AT N5 OR N10 • FORMIMINO (-CH=NH) AT N5 • METHENYL ( -CH=) AT N5,N10 • LOOK AGAIN AT THE 2 REACTIONS FOR SYNTHESIS OF GLY • SERINE HYDROXYMETHYLTRANSFERASE • GLYCINE SYNTHASE • THF IS INVOLVED IN EACH

30. TETRAHYDROFOLATE • C1 UNITS ENTER THE THF POOL MAINLY FROM THESE TWO REACTIONS • AS N5,N10 –METHYLENE-THF OXIDATION STATES OF C1 UNITS ATTACHED TO THF ARE INTERCONVERTIBLE VIA ENZYMATIC REDOX REACTIONS • WE WILL SEE THF AGAIN • METHIONINE SYNTHESIS • HIS SYNTHESIS • PURINE SYNTHESIS • dTMP (THYMIDYLATE) SYNTHESIS

31. TETRAHYDROFOLATE • THF IS DERIVED FROM FOLIC ACID • MAMMALS CANNOT SYNTHESIZE IT • DEFICIENCY DURING EARLY PREGNANCY CAN LEAD TO NEURAL TUBE DEFECTS • ANENCEPHALY   SPINA BIFIDA • BACTERIA SYNTHESIZE FOLIC ACID • SULFONAMIDES COMPETITIVELY INHIBIT • STRUCTURAL ANALOGS OF PABA • GOOD ANTIBACTERIAL AGENTS • WHY ARE MAMMALS UNAFFECTED?

32. TETRAHYDROFOLATE • STUDY QUESTION: IF I GIVE YOU THE STRUCTURE OF THF, NUMBERING THE ATOMS ACCORDINGLY, BE ABLE TO SHOW WHERE TO ATTACH THE 5 DIFFERENT C1 GROUPS.

33. TRANSAMINATION REACTIONSIN-CLASS STUDY QUESTION • DRAW THE STRUCTURES OF THE KETO-ACID PRODUCTS OF THE REACTIONS OF THE FOLLOWING AMINO ACIDS WITH -KG. • GLY • ARG • SER • DRAW THE STRUCTURE OF THE AMINO ACID PRODUCT COMMON TO ALL 3 RXNS’

34. REFERENCES • HERE ARE TWO ARTICLES THAT MIGHT HELP YOU TO ORGANIZE YOUR THINKING ABOUT AMINO ACID METABOLISM: (1) “Glutamate and Glutamine, at the Interface between Amino Acid and Carbohydrate Metabolism” (Brosnan JT, The Journal of Nutrition, Apr 2000, 130,4S: 988S – 990S) (2) “Disorders of Glutamate Metabolism” (Kelly A, Stanley CA, 2001. Mental Retardation and Developmental Disabilities Research Reviews, 7:287-295

35. SYNTHESIS OF ESSENTIAL AMINO ACIDS • ALL SYNTHESIZED FROM COMMON METABOLIC PRECURSORS • ASPARTATE • PYRUVATE • PHOSPHOENOLPYRUVATE • ERYTHROSE-4-PHOSPHATE • PURINE + ATP (HISTIDINE) • PATHWAYS ONLY IN MICRO-ORGANISMS AND PLANTS • PROBABLE EVOLUTIONARY LOSS IN MAMMALS • PATHWAYS ARE VERY COMPLICATED • ACTUAL PATHWAYS VARY ACROSS SPECIES! • IN CONTRAST TO LIPID AND CARBOHYDRATE PATHWAYS, WHICH ARE ALMOST UNIVERSAL

36. ESSENTIAL AMINO ACID SYNTHESIS • FOUR “FAMILIES” • ASPARTATE • LYS • MET • THR • PYRUVATE • LEU, ILE, VAL (THE “BRANCHED CHAIN” AMINO ACIDS) • AROMATIC • PHE • TYR • TRP • HISTIDINE

37. THE ASPARTATE FAMILY • FIRST COMMITTED STEP IS • ASP + ATP  ASPARTYL-β-PHOSPHATE + ADP • ENZYME: ASPARTOKINASE • 3 ISOZYMES IN E.coli • EACH RESPONDS DIFFERENTLY AS FAR AS FEEDBACK INHIBITION AND REPRESSION OF ENZYME SYNTHESIS • THR,LYS, MET PATHWAYS INDEPENDENTLY CONTROLLED

38. THE ASPARTATE FAMILY • CONTROL OF ASPARTOKINASE ISOENZYMES • ENZYME FEEDBACK INHIB COREPRESSOR ASP I THR THR, ILE ASP II NONE MET ASP III LYS LYS • COREPRESSOR: TRANSCRIPTIONAL REPRESSION

39. ASPARTATE FAMILY • ALSO CONTROL AT BRANCH POINTS • NOTE THE FOLLOWING REACTION: • HOMOCYSTEINE + N5-METHYL-THF  MET + THF • ENZYME: METHIONINE SYNTHASE (?)  HOMOCYSTEINE  CV DISEASE RISK FACTOR • EAT FOODS CONTAINING FOLATE • RECALL:SER + HOMOCYSTEINE  CYSTATHIONINE • ENZYME DEFECTS IN REMETHYLATION OF HOMOCYSTEINE TO MET OR IN RXN’ FROM CYSTATHIONINE  CYS  HOMOCYSTEINE • DEFECT IN SYNTHESIS OF CYSTATHIONE-β-SYNTHASE • HYPER HOMOCYSTENEMIA  HOMOCYSTEINURIA • SYMPTOMS: • PREMATURE ATHEROSCLEROSIS • THROMBOEMBOLIC COMPLICATIONS • SKELETAL ABNORMALITIES • ECTOPIA LENTIS • MENTAL RETARDATION

40. THE PYRUVATE FAMILY • “BRANCHED CHAIN AMINO ACIDS” • LEU • ILE • VAL • VAL, ILE: SAME PATHWAY AFTER 1st STEP • LEU PATHWAY BRANCHES FROM VAL PATHWAY • FINAL STEPS ALL CATALYZED BY AMINO- TRANSFERASES • GLU IS THE AMINO DONOR

41. THE PYRUVATE FAMILY • THE FIRST STEP: • PYR + TPP  HYDROXYETHYL-TPP • FIRST PYR AND TPP FORM AN ADDUCT • THEN DECARBOXYLATED TO HE-TPP • A RESONANCE-STABILIZED CARBANION • A STRONG NUCLEOPHILE • ADDS TO KETO GROUP OF • PYRUVATE  VAL, LEU • -KETOBUTYRATE  ILE

42. THE PYRUVATE FAMILY • LOOK AT THE REACTION MECHANISM OF PYRUVATE DECARBOXYLASE (PAGE 605) • THIS SHOWS THE FORMATION OF THE HYDROXYETHYL-TPP ADDUCT • THIAMINE (VIT B1) • SOME INTERESTING CHEMISTRY • THIAZOLIUM RING • ACIDIC HYDROGEN • “ELECTRON SINK” • TRANSITION STATE STABILIZATION MECH. • YLIDS • RESONANCE

43. THE AROMATIC FAMILY • IN PLANTS AND MICRORGANISMS • PHE • TYR • TRP • PECURSORS ARE: • PEP • ERYTHROSE-4-PHOSPHATE • THESE CONDENSE WITH ULTIMATE CONVERSION TO CHORISMATE

44. THE AROMATIC FAMILY • CHORISMATE • BRANCH POINT FOR TRP SYNTHESIS • CHORISMATE ANTHRANILATE TRP • CHORISMATE  PREPHENATE • PREPHENATE • BRANCH POINT FOR PHE, TYR SYNTH • AMINOTRANSFERASES IN EACH FINAL STEP • IN MAMMALS, TYR IS A PRODUCT OF: • PHE HYDROXYLATION

45. THE TRP PATHWAY • TRYPTOPHAN SYNTHASE • CATALYZES FINAL 2 STEPS INDOLE-3-GLYCEROL PHOS  INDOLE + GLYC-3-P INDOLE + SER  H2O + TRP • 2β2 BIFUNCTIONAL ENZYME • WHAT ENZYME CLASS?

46. THE TRP PATHWAY • “CHANNELING” • INDOLE IS SEQUESTERED BETWEEN THE TWO ACTIVE SITES • DIFFUSES BETWEEN TWO SITES • IT’S NONPOLAR • STUDY QUESTION: • WHAT ARE THE BENEFITS OF CHANNELING? • SEE RIBBON DIAGRAM OF TRP SYNTHASE ON PAGE 1044 • MECHANISM?

47. PHENYLKETONURIA (PKU) • DEFECTIVE OR ABSENT PHENYLALANINE HYDROXYLASE CANNOT FORM TYROSINE PHE BUILDS UP •  PHE IS TRANSAMINATED TO PHENYL-PYRUVATE • SEVERE MR IF NOT TREATED SOON AFTER BIRTH WITH LOW PHE DIET • UNIVERSAL NEWBORN SCREENING

48. PHENYLKETONURIAIN-CLASS STUDY QUESTION • WRITE OUT THE REACTION IN WHICH PHE IS TRANSAMINATED TO PHENYLPYRUVATE, SHOWING STRUCTURES • EXPLAIN WHY CHILDREN WITH A TETRAHYDRO-BIOPTERIN DEFICIENCY EXCRETE LARGE AMOUNTS OF PHE • WHY DO PEOPLE WITH PKU HAVE BLOND HAIR, BLUE EYES AND VERY LIGHT SKIN? • WHY DO PEOPLE ON A LOW PHE-DIET NEED TO INCREASE THEIR TYR INTAKE?

49. HISTIDINE BIOSYNTHESIS • ATOMS DERIVED FROM: • 5-PHOSPHORIBOSYL--PYROPHOSPHATE • PROVIDES 5 C-ATOMS • PRPP INVOLVED IN PURINE SYNTHESIS • PRPP INVOLVED IN PYRIMIDINE SYNTHESIS • PURINE SALVAGE PATHWAY • AN INTERMEDIATE IN TRP SYNTHESIS • ATP PROVIDES THE 6th C-ATOM • ATP + -D-RIBOSE-5-PHOSPHATE  PRPP + AMP • -D-RIBOSE-5-PHOSPHATE FROM H-M SHUNT

50. HISTIDINE BIOSYNTHESIS • NOTICE THE PRODUCTS OF THE AMIDO-TRANSFERASE STEP: • AICAR • AN INTERMEDIATE IN PURINE BIOSYNTHESIS • IMIDAZOLE GLYCEROL PHOSPHATE • THERE IS AN APPARENT EVOLUTIONARY OVERLAP OF PURINE AND HIS SYNTHESIS • THE FIRST STEP IN HIS SYNTHESIS INVOLVES FORMATION OF A PURINE!