1. 1 Part 3 Coenzymes-Dependent Enzyme Mechanism Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are not my own, they can be found on: –References supplied –Atlases or –The web King Saud University College of Science Department of Biochemistry

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3. 3 Mechanism of Carbanion Stabilization by PLP

4. 4  -Amino Acid  Internal aldimine ( PLP-Enz Schiff base) External aldimine (PLP-substrate Schiff base)

5. 5 Mechanism of Carbanion Stabilization by PLP, Cont’d  Stabilized carbanion resonance

6. 6 Mechanism of Carbanion Stabilization by PLP, Cont’d    Stabilized carbanion resonance

7. 7 Mechanism of Carbanion Stabilization by PLP, Cont’d    Stabilized carbanion resonance

8. 8 Mechanism of Carbanion Stabilization by PLP, Cont’d   Stabilized carbanion resonance

9. 9 Mechanism of Carbanion Stabilization by PLP, Cont’d   Stabilized carbanion resonance

10. 10 Mechanism of Carbanion Stabilization by PLP, Cont’d   Stabilized carbanion resonance For determination of stereochemistry of amino acid formed

11. 11 Mechanism of Carbanion Stabilization by PLP, Cont’d     Stabilized carbanion resonance

12. 12 Jencks’ Statement The versatile chemistry of pyridoxal phosphate offers a rich learning experience for the student of mechanistic chemistry Professor W. Jencks, in his classic text, Catalysis in Chemistry and Enzymology, writes: –“ It has been said that God created an organism especially adapted to help the biologist find an answer to every question about the physiology of living systems;

13. 13 –if this is so it must be concluded that pyridoxal phosphate was created to provide satisfaction and enlightenment to those enzymologists and chemists who enjoy pushing electrons, for no other coenzyme is involved in such a wide variety of reactions, in both enzyme and model systems, which can be reasonably interpreted in terms of the chemical properties of the coenzyme Jencks’ Statement, Cont’d

14. 14 –Most of these reactions are made possible by a common structural feature –That is, electron withdrawal toward the cationic nitrogen atom of the imine and into the electron sink of the pyridoxal ring from the a carbon atom of the attached amino acid activates all three of the substituents on this carbon atom for reactions which require electron withdrawal from this atom” * * Jencks, William P., 1969. Catalysis in Chemistry and Enzymology. New York: McGraw-Hill Jencks’ Statement, Cont’d

15. 15 Biochemical Functions of Pyridoxal phosphate 1.Decarboxylation of amino acids 2.Transaminase reactions 3.Racemization reactions 4.Aldol cleavage reactions 5.Transulfuration reactions 6.Conversion of tryptophan to niacin 7.Conversion of linoleic acid into arachidonic acid (prostaglandin precursor) 8.Formation of sphingolipids

16. 16 Transamination Reactions

17. 17 The term transamination refers to the interconversion of carbonyl and amino groups. Condensation of an amine with an aldehyde as shown below gives an imine. What is formally only a tautomerisation reaction converts imine A into its tautomer B which upon hydrolysis yields the "transaminated" products. i.e. the product in which the amine and the carbonyl group have been swapped..

18. 18 This is a highly simplified view of the transamination reaction. Firstly, aldehydes do not occur in biological systems due to their chemical instability. The biological equivalent of aldehydes are imines.

19. 19 Firstly, aldehydes do not occur in biological systems due to their chemical instability. The biological equivalent of aldehydes are imines.

20. 20 Secondly, imines are chemically stable towards this type of tautomerisation reaction. An enzyme is required to effect this transformation and the enzymes employs a co-factor (or prosthetic group).This cofactor is pyridoxal phosphate (PLP).

21. 21 PLP is attached to the enzyme forms an imine with a lysine residue. This link attaches the co-factor to the enzyme and converts the aldehyde into its biological equivalent, the imine. The conversion of amino acids into a-keto acids (also sometimes referred to as a-oxo- acids) is a central reaction of primary and secondary metabolism.

22. 22 In the first step of transamination reactions, pyridoxalphosphate in its biological form of imine is tranferred to the substrate amino acid.

23. 23

24. 24 Then the PLP-dependent enzymes catalyses the tautomerisation of the imime.

25. 25 In the final step, hydrolysis of the imine gives the products. Note, that pyridoxal phosphate (PLP) has been converted into pyridoxamine by the transamination reaction. A second transamination step is required to convert pyridoxamine back into PLP. This restores the co-factor and the enzyme can carry out another transamination reaction.

26. 26 Mechanism of PLP-catalysed transaminations The a-hydrogen of the imine is in conjugation with the protonated pyridinium nitrogen. The positively chareged nitrogen increases the aciditiy of the a-hydrogen and facilitates proton abstraction. The product is an extended conjugated system incorporating both an imine and an enamine.

27. 27

28. 28 In the final step, protonation occurs at the d-carbon to the pyridine nitrogen, thus restoring the aromatic system. Hydrolysis of the imine gives the final products.

29. 29 Decarboxylation and PLP Decarboxylation reactions are important in biological systems because intermediates which are chemically disposed for decarboxylation, such as b-keto acids, occur frequently in primary and secondary metabolism.

30. 30 a-Keto acids are chemically not predisposed towards decarboxylation. This is reflected in much higher temperatures required to effect the above transformation. Nature uses enzymes for this reaction which carry PLP as co-factor. The schemes below shows the decarboxylation of an a-amino acid.

31. 31 The amino acid is bound to to PLP as the imine in the first step.

32. 32 In the actual decarboxylation step, the electronic effects are the same: the pyridine nitrogen acts as an electron- withdrawing group, this time facilitating deprotonation of the carboxylic acid group. Loss of carbondioxide and hydrolysis of the imine gives the reaction products.

33. 33 Transamination Reactions  -Amino acid  -Keto acid  -Amino acid

34. 34 Transamination Reactions Most common amino acids can be converted into the corresponding keto acid by transamination This reaction swaps the amino group from one amino acid to a different keto acid, thereby generating a new pairing of amino acid and keto acid There is no overall loss or gain of nitrogen from the system

35. 35 Transamination reactions are readily reversible, and the equilibrium constant is close to 1 One of the two substrate pairs is usually Glu and its corresponding keto acid  -KG Transamination Reactions, Cont’d

36. 36 The effect of transamination reactions is to collect the amino groups from many different amino acids in the form of L-Glu The Glu then functions as an amino group donor for biosynthetic pathways or for excretion pathways that lead to the elimination of nitrogenous waste products Transamination Reactions, Cont’d

37. 37 The substrates bind to the enzyme active center one at a time, and the function of the pyridoxal phosphate is to act as a temporary store of amino groups until the next substrate comes along In the process the pyridoxal phosphate is converted into pyridoxamine phosphate, and then back again Enzymologists call this a ping pong mechanism Transamination Reactions, Cont’d

38. 38 The condensation between the  -amino group and the aromatic aldehyde to form a Schiff base makes the  -carbon atom chemically reactive, so the isomerization of the Schiff base takes place very easily Many of the enyzmes that metabolize amino acids require PLP as a cofactor Unexpectedly, this compound also serves in a completely different manner in the active center of glycogen phosphorylase Transamination Reactions, Cont’d

39. 39 Comparison of the active sites of L- aspartate aminotransferase (left) and D-amino acid aminotransferase (right)

40. 40 The three-dimensional structures of bacterial D-amino acid aminotransferase (top) and human mitochondrial branched- chain L-amino acid aminotransferase (bottom)

41. 41 Aspartate Transaminase (Aspartate Aminotransferase)

42. 42 Aspartate Transaminase Aspartate transaminase (AST) also called serum glutamic oxaloacetic transaminase (SGOT) or aspartate aminotransferase (ASAT/AAT) (EC 2.6.1.1) is similar to alanine transaminase (ALT) in that it is another enzyme associated with liver parenchymal cells PLP coenzyme provides an aldehyde group to the enzyme, which is not available among the side chains of the 20 amino acids found in proteins

43. 43 The phosphate group provides a way to bind the coenzyme to the enzyme via a strong ionic interaction The aldehyde group readily reacts with primary amines like the  -amino groups of amino acids This process activates the amino group so that it can be cleaved by water Aspartate Transaminase, Cont’d

44. 44 This releases the keto-acid core of the amino acid and leaves the amino group on the enzyme Now the acceptor keto-acid binds and reacts with the activated amino group to form the new amino acid Aspartate Transaminase, Cont’d

45. 45 Aspartate Transaminase, Cont’d The mitochondrial aspartate transaminase provides an especially well studied example of PLP as a coenzyme for the transamination reactions The results of X-ray crystallographic studies provided detailed views of how PLP and substrates are bound and confirmed much of the proposed catalytic mechanism

46. 46 Aspartate Transaminase, Cont’d The enzyme is a dimer if identical subunits and it consists of a large domain and a small one PLP is bound to the large domain, in a pocket near the subunit interface In the absence of substrate, the aldehyde group of PLP is in a Schiff base linkage with Lys-258 Arg-386 interacts with the  -carboxylate group of the substrate, helping to orient the substrate appropriately in the active site

47. 47 Structure of Aspartate Transaminase The active site of enzyme includes PLP attached to the enzyme by Schiff base linkage with Lys-258 Arg-386 residue in the active site helps orient substrates by binding to their  -carboxylate groups

48. 48 Structure of Aspartate Transaminase Schematic diagram of the active site of E. coli aspartate aminotransferase Substrate specificity for the negatively charged aspartic acid substrate is determined by the positively charged guanidino groups of Arg-386 and Arg-292, which have no catalytic role Mutation of Arg-292 to Asp produces an enzyme that prefers Arg to Asp as a substrate

49. 49 Stereochemistry of Aspartate Transaminase Reaction PLP enzymes cleave one of three bonds at the C  atom of amino acids For example, bond a is cleaved by aminotransferase, bond b by dehydrogenase, and bond c by aldolase How can the same amino acid-PLP Schiff base be involved in the cleavage of the different bonds to an amino acid C 

50. 50 Stereochemistry of Aspartate Transaminase Reaction, Cont’d For electrons to be withdrawn into the conjugated ring system of PLP, the  -orbital system of PLP must overlap with the bonding orbital containing the electron pair being delocalized This is possible only if the bond being broken lies in the plane perpendicular to the plane of the PLP  -orbital system Different bonds to C  can be placed in this plane by rotation about the C  -N bond

51. 51 Stereochemistry of Aspartate Transaminase Reaction, Cont’d Each enzyme specifically cleaves its corresponding bond because the enzyme binds the amino acid-PLP Schiff base adduct with this bond in the plane perpendicular to that of the PLP ring This is an example of stereoelectronic assistance (effect) The enzyme binds substrate in a conformation that minimizes the electronic energy of the transition state

52. 52 Stereochemistry of Aspartate Transaminase Reaction, Cont’d Bond orientation in a PLP–amino acid Schiff base The  -orbital framework of a PLP–amino acid Schiff base The bond to C  in the plane perpendicular to the PLP  -orbital system

53. 53 In PLP-dependent transaminase’s active site, the addition of H + from Lys residue to the bottom face of the quinoid intermediate determines the L- configuration of the amino acid product The conserved Arg residue interacts with the  - carboxylate group and helps establish the appropriate geometry of the quinonid intermediate Stereochemistry of Aspartate Transaminase Reaction, Cont’d

54. 54 Mechanism of L-Configuration of Amino Acids Produced   Stabilized carbanion resonance For determination of stereochemistry of amino acid formed

55. 55 The orientation about the NH-C  bond determines the most favored reaction catalyzed by PLP- dependent enzymes The bond that is most nearly perpendicular to the  orbital of the PLP electron sink is most easily cleaved Stereochemistry of Aspartate Transaminase Reaction, Cont’d

56. 56 In PLP-dependent transaminases, C  -H bond is most nearly perpendicular to the  orbital system and is cleaved In SHMT, a small rotation about N-C  bond places the C  -C  bond perpendicular to the  system, favoring its cleavage Stereochemistry of Aspartate Transaminase Reaction, Cont’d

57. 57 Mechanism of Aspartate Transaminase

58. 58 Reaction of Aspartate Transaminase L-Asp L-Glu OAA  -KG Asp Transaminase

59. 59 Reaction of Aspartate Transaminase L-Asp OAA  -KG L-Glu Ping Pong Mechanism E-PLPPLP-Asp PLP-OAAPLP-a-KG PLP-GluE-PMPE-PLP

60. 60 Active Site of Asp Transaminase External aldimine (PLP-Asp Schiff base) General base Both carboxylate groups of Asp are bound by electrostatic interactions to the active site Arg-292 and Arg-386

61. 61 Mechanism of Asp Transaminase PLP Tetrahedral intermediate

62. 62 Mechanism of Asp Transaminase, Cont’d PLP-Enzyme Schiff base (Enzyme aldimine) Asp

63. 63 Mechanism of Asp Transaminase, Cont’d Tetrahedral intermediate

64. 64 Mechanism of Asp Transaminase, Cont’d PLP-Asp Schiff base (Asp aldimine) Quinonoid Abstract  -carbon

65. 65 Mechanism of Asp Transaminase, Cont’d KitimineTetrahedral intermediate OAA

66. 66 Mechanism of Asp Transaminase, Cont’d PMP Tetrahedral intermediate  -KG

67. 67 Mechanism of Asp Transaminase, Cont’d Protonation at  -carbon Kitimine Glu aldimine

68. 68 Mechanism of Asp Transaminase, Cont’d Enzyme aldimine (PLP-Enzyme Schiff base) Glu Tetrahedral intermediate

69. 69 By using site-directed mutagenesis techniques by replacing Lys-258 for Ala gives a completely inactive mutant enzyme Replacing Lys-258 for Cys, the mutant enzyme is similarly inactive, however, if this enzyme is alkylated with 2-bromoethylalanine an active enzyme is obtained which contains a thioether analog of Lys at the active site Experimental Evidences for the Role of Lys-258, Arg-385 and Arg- 292

70. 70 This enzyme has 7% of the activity of wild- type enzyme with a slightly shifted pH rate profile of enzymatic activity Since the thioether-containing Lys analog is slightly less basic than Lys By replacing Arg-292 by other amino acids, mutation of Arg-292 to Asp-292 gave an enzyme whose catalytic efficiency for L-Asp has dropped from 34500 to 0.07 M -1 s -1 Experimental Evidences for the Role of Lys-258, Arg-385 and Arg- 292, Cont’d

71. 71 Experimental Evidences for the Role of Lys-258, Arg-385 and Arg- 292, Cont’d However, mutant enzyme was found to be capable of processing L-amino acid substrates containing positively charged side chains (Arg, Lys, and ornithine) which would interact favorably with Asp- 292 with kcat/Km of 0.43 M -1 s -1