1

Chapter One

General Introduction
 2

A Brief History of Vitamin B6

Early research performed in the 1920’s had identified two major components of

the vitamin B complex: vitamin B1; the anti-neuritic factor and vitamin B2; the

antipellagra factor.1, 2 Further work on vitamin B2 yielded individual “factors” named

vitamin B3, B4 and B51 with the rat pellagra factor named vitamin B6 in 1934.3 Vitamin

B6 was isolated in a pure crystalline form in 1938 and in 1939 the structure of a vitamin

B6 derivative was determined and the name pyridoxine (Figure 1.1) was first proposed.1, 2

In the 1940’s two other active forms of vitamin B6; pyridoxal (Figure 1.1) and

pyridoxamine (Figure 1.1) were discovered by Snell.4 By the mid-1940’s the structure of

the fully active cofactor pyridoxal 5'-phosphate (PLP) (Figure 1.1) was determined and

the first synthesis of PLP from pyridoxine was performed.5 By the early 1950’s the role

of PLP in enzymatic transamination, racemization and β-elimination had been

established4 and the structural features essential for enzymatic and non-enzymatic

vitamin B6 catalyzed reactions were being elucidated. This culminated in a 1954 article

proposing a “general mechanism for vitamin B-6 catalyzed reactions.”6 This article,

which was cited 794 times over the last 56 years,7 lays out the central structural

requirements of vitamin B6 catalysis that have become widely accepted over the last half

century. These include an aldehyde at C4', a phenol at C3' and the pyridine ring nitrogen,

N1 (see Figure 1.1 for numbering scheme). Notably, the role of the protonated ring

nitrogen acting as an electron sink in carbanion stabilization (Figure 1.2-“quinonoid”)

was proposed in this seminal work and has since become the hallmark of PLP catalyzed

reactions; invoked in nearly every mechanism presented in both journal articles and

textbooks.8-11 By 1960, PLP was seen as “hold[ing] an exceptional place among the
 3

coenzymes with regard both to the unparalleled diversity of its catalytic function and to

[its] paramount significance in biochemical transformations of amino acids…”.12 Indeed,

the wide variety of reactions catalyzed by PLP is impressive, including; decarboxylation,

racemization, transamination, β- elimination, retro Aldol cleavage and others.8, 9

Complementing this diversity are the common mechanistic themes provided by a general

mechanism. As noted by D.E. Metzler, 32 years after first proposing the general

mechanism: “People like the fact that so many different enzymatic reactions can be

understood from the underlying properties of the coenzyme.”13 To date over 140 enzyme

commission entries (4% of total) are PLP dependent and it is estimated that prokaryotes

devote ~1.5% of their open-reading frames to PLP dependent enzymes.14 Given the wide

variety of reactions catalyzed by PLP, the large number of PLP utilizing enzymes and the

satisfaction of unifying mechanistic themes, PLP has been among the most studied

cofactors for over half of a century.

 4

Reactivity and Specificity of PLP Utilizing Enzymes

Despite the wide variety of PLP catalyzed reactions, as mentioned in the above

section, unifying mechanistic themes exist. In the absence of substrate, all PLP enzymes

exist in a Schiff base linkage, termed the “internal aldimine”, between the C4' aldehyde

of the PLP cofactor and the ε-amine of an active site lysine residue (Figure 1.2). Upon

introduction of substrate, typically an amino acid, the active site lysine residue is

displaced in a process termed transimination. The new Schiff base linkage between the

amine group of the substrate and the C4' aldehyde of the cofactor is termed the “external

aldimine” (Figure 1.2). All subsequent reactions proceed from the external aldimine9

(Figure 1.2). The great majority of reactions proceed by a carbanionic intermediate

formed by cleavage of one of the three bonds to Cα of the substrate.8, 9, 15 Loss of the Cα

proton, required for transamination, racemization and β-elimination, is the most common

route to carbanion formation (Figure 1.2). Stabilization of the resulting carbanion,

regardless of which Cα group is cleaved, is largely thought to occur through resonance

delocalization with the conjugated π electron system of the cofactor (Figure 1.2-

resonance forms). The electron sink properties of PLP in carbanion stabilization, as

exemplified in “quinonoid” formation (Figure 1.2) is thought to be largely responsible for

the catalytic prowess of PLP.9 Two points regarding the mechanistic themes described

above and shown in Figure 1.2 require further discussion. First, how do enzymes

selectively cleave a particular Cα bond when presented with three possibilities? Second,

how does the enzyme control the fate of the quinonoid intermediate; i.e. in the case of

cleaving the Cα proton, how is racemization versus transamination enforced? In 1966

Dunathan proposed a mechanism through which PLP utilizing enzymes could select for
 5

the Cα group to be cleaved and also labilize the selected Cα σ bond through

hyperconjugation with the extended π electron system of the PLP cofactor16 (Figure 1.3).

Orientation of the bond to be cleaved orthogonal to the plane of the cofactor aromatic

ring allows for maximum orbital overlap between the bond of the Cα group and the π

electron system of the cofactor. This hyperconjugation “activates” the Cα σ bond for

cleavage through two possible means: 1) ground state destabilization of the σ bond upon

binding of substrate and 2) resonance stabilization of the negative charge in the

unhybridized p orbital formed upon cleavage. That PLP-dependent enzymes bind

substrate in accordance with Dunathan’s hypothesis is documented by X-ray

crystallography studies of external aldimines formed with unreactive substrate

analogues,17, 18 as shown in Figure 1.4. While Dunathan’s hypothesis provides a means

by which enzymes may selectively labilize the Cα bond to be cleaved in carbanion

formation, the subsequent fate of the carbanion needs further analysis. As shown in

Figure 1.2, cleavage of the Cα hydrogen is not limited to a single reaction type. The

interconversion of D and L-alanine by alanine racemase provides a nice case study

(Figure 1.6). Upon formation of the carbanion intermediate several possible reactions are

possible: 1) reprotonation of the Cα carbon on the same face with no racemization, 2)

reprotonation from the opposite face resulting in stereocenter interconversion and 3)

reprotonation at C4' of the cofactor by the repositioning of either Lys39 or Tyr265

leading to transamination.9 Alanine racemase is able to severely suppress transamination,

with only one occurrence for ~106 racemization reactions.19 By maintaining the pyridine

ring nitrogen of PLP in an unprotonated state8, 9 the quinonoid intermediate is selectively

destabilized resulting in a shorter lifetime. This prevents the slower (relative to

 6

racemization) repositioning of Lys39 or Tyr265, leading to transamination.9 Thus a

combination of controlling the protonation state of the pyridine ring nitrogen and tight

regulation of the positioning of active site residues provide alanine racemase a means to

achieve high reaction fidelity.

 7

Structural Relationships of PLP Utilizing Enzymes

All PLP utilizing enzymes with known three dimensional structures fall into one

of five families, or fold types. These families, based upon sequence alignments and

three-dimensional structural information, are not limited to a single reaction type.8, 20 The

structural features common to all five families include binding of the 5'-phosphate group

to the N terminus of an α helix and covalent linkage of the PLP cofactor with an active

site residue via a Schiff base.20

Fold Type I: the aspartate aminotransferase family. This family contains the

greatest number of known structures and is therefore the best characterized of the five

fold types. Enzymes in this family largely exist as homodimers (some contain larger

complexes) with each subunit containing a small and large domain. The enzyme active

site is located in a cleft found between the two domains and is made up of residues from

both domains and both subunits. All members of this class contain a conserved aspartic

acid residue that interacts with the pyridine nitrogen of PLP, thus fully activating the

electron sink capabilities of the cofactor.

Fold Type II: the tryptophan synthase family. As with the aspartate

aminotransferae family, the enzymes exist as homodimers or higher order oligemers but

the active sites are made up from residues from a single monomer. Additionally,

enzymes of this class usually contain regulatory domains.20 The pyridine ring nitrogen of

the cofactor is hydrogen bound to an active site serine in this family and is not expected

to be protonated.

Fold Type III: the alanine racemase family. These enzymes are dimers, with the

active site made up from residues from each monomer. The structural motifs are quite
 8

different from the above fold types, having a classical TIM barrel. Despite this, the

binding of PLP is very similar to the other families. The pyridine ring nitrogen is

hydrogen bound with an arginine residue and, as with the tryptophan synthase family, is

not expected to be protonated.

Fold Type IV: D-amino acid aminotransferase family. These enzymes exist as

homodimers with each monomer being made up of a large and small domain. The active

site, located at the domain interface is a mirror image of the aspartate aminotransferase

family active site, with the re face of the cofactor facing the protein as opposed to

solvent. A glutamic acid residue protonates the pyridine ring nitrogen.

Fold Type V: the glycogen phosphorylase family. As with the alanine racemase

family, the structure of this family differs greatly from the other fold types. PLP does not

act in the usual manner as seen with fold types I-IV. Instead, the phosphate moiety is

involved in proton transfer. Despite this, PLP is required for catalysis and this enzyme is

included in the super family of PLP utilizing enzymes.

 9

Escherichia coli Aspartate Aminotransferase

Aspartate aminotransferase, the prototypical fold type I enzyme, binds PLP in an

active site located at the subunit interface, comprised of amino acids from residues of

both subunits. The PLP cofactor is tightly bound in the active site forming a Schiff base

with lysine 258 and nine hydrogen bonds to the phosphate moiety. Additional

interactions include: the binding of O3' position by tyrosine 225 and asparagine 194; π

stacking with tryptophan 140 on the si face and protonation of the pyridine ring nitrogen

through a salt bridge with aspartate 222.

AATase catalyzes the reversible 1, 3-prototropic shift between C4' of the PLP

cofactor and the Cα position of dicarboxylic amino acids and their corresponding α-keto

acids via a ping-pong bi-bi mechanism (Figure 1.5) thus playing a vital role in the

intermolecular transfer of nitrogen between its substrates. In the absence of substrate

PLP exists in a Schiff base with the ε-amino group of lysine 258. In this form of the

internal aldimine the Schiff base nitrogen has a pKa of ~721 which absorbs maximally at

either ~430 nm (protonated form: ε430 nm = 8700 M-1 cm-1)22 and 360 nm (unprotonated

form). Upon introduction of substrate the amino group of the amino acid displaces lysine

258, forming a new Schiff base termed the external aldimine. Transimination is

accompanied by a conformational change from the open form of the internal aldimine to

a closed form in the external aldimine which shields the re face of the cofactor from

exposure to solvent minimizing the possibility of racemization. This occurs with an

increase in the pKa of the Schiff base nitrogen in the external aldimine to a value greater

than 10, displaying maximal absorbance solely at ~430 nm. Concomitant with

transimination is a tilt of the cofactor towards substrate by ~17° relative to the internal
 10

aldimine.17, 21 The carboxylate groups of the substrate interact with the side chains of

arginines 286 and 392, holding the Cα proton perpendicular to the plane of the cofactor

ring (Figure 1.4). This orientation of the substrate selectively labilizes the Cα proton in

accordance with Dunathan’s hypothesis.16 Upon deprotonation of the external aldimine,

the largely rate determining step, the carbanionic intermediate, or “quinonoid” is

reprotonated at the C4' position to yield the ketimine. The quinonoid intermediate may

be directly observed in AATase catalyzed reactions as a peak at ~500 nm with high

extinction coefficient.9 Hydrolysis of the ketimine yields the α-keto acid of the amino

acid substrate and pyridoxamine 5'-phosphate, with a maximum absorbance at ~330 nm

(ε325 nm = 7300 M-1, cm-1).23 The PLP internal aldimine is regenerated by a reversal of

the above reactions with a second α-keto acid. The spectral changes accompanying

conversion of the external aldimine (430 nm) to PMP (330 nm) allows for direct UV-vis

monitoring of the first half reaction by the disappearance of the external aldimine or the

appearance of PMP.
 11

Bacillus stearothermophilus Alanine Racemase

Alanine racemase, a fold type III enzyme, exists as a homodimer, with two active

sites located at the monomer interface containing residues from each monomer.

Interactions with the PLP cofactor and the active site include: the Schiff base linkage

between C4' of PLP and lysine 39; fixing the 5' phosphate of PLP in place by hydrogen

bonds to tyrosine 43, serine 204, and tyrosine 354; hydrogen bonding between the O3'

phenol of the cofactor and arginine 136; and hydrogen bonding between the pyridine

nitrogen of PLP and arginine 219.24 Given the pKa difference between arginine 219

(~13) and the pyridine ring nitrogen (~5), one would not expect protonation of the

pyridine ring nitrogen. Additionally, tyrosine 265 from the other monomer comprising

the active site is postulated to serve as a general acid/base in racemization.25

Alanine racemase catalyzes the interconversion of the L and D isomers of alanine

(Figure 1.6), thus making it an essential enzyme in the biosynthesis of cell walls. As with

all PLP enzymes, the resting state is an internal aldimine (ε420 nm = 8450 M-1 cm-1).26

Upon introduction of either L or D alanine the enzyme undergoes transimination, forming

the external aldimine, thus displacing lysine 39. The next step, Cα proton abstraction,

followed by reprotonation at the opposite face of substrate provides the isomerized

product. Tyrosine 265 and lysine 39 serve as the general acid/base catalysts for

racemization: tyrosine 265 as the base and lysine 39 as the acid for L to D direction while

in the D to L direction their respective roles are reversed. Initially, the lack of a directly

observable quinonoid intermediate with the wild type enzyme presented the possibility of

a concerted mechanism by which the carbanionic intermediate (quinonoid) may be

avoided. However, it has been determined through kinetic isotope effects that the
 12

reaction is indeed stepwise with a high energy quinonoid present in the mechanism.27

Mutation of arginine 219 to glutamate allows for direct observation of the buildup of the

quinonoid intermediate,25 which is consistent with the contention that a protonated

pyridine ring nitrogen is a requirement for this intermediate. Interestingly, it is precisely

the unstable quinonoid intermediate which may be responsible for the high fidelity seen

with alanine racemase. Transamination, which likely requires a rate determining

movement of the active site lysine or tyrosine to protonate the C4' carbon of PLP, would

be suppressed by the short lifetime of the quinonoid intermediate.9 Unlike aspartate

aminotransferase and O-acetylserine sulfhydrylase (below), there are no spectral changes

associated with racemization of alanine that allow for direct UV-vis monitoring.
 13

Salmonella typhimurium O-Acetylserine Sulfhydrylase Isozyme A

O-Acetylserine sulfhydrylase A (OASS) is a fold type II enzyme having an overall

structure similar to the β2 subunit of tryptophan synthase.28 OASS exists as a homo

dimer with each monomer being composed of two domains. Each monomer binds one

PLP molecule in a Schiff base with lysine 41 located deep within the protein, at the

interface of the two domains. The 5' phosphate of PLP anchors the cofactor in the active

site, through hydrogen bond interactions with a glycine and threonine loop. The O3'

phenol of the cofactor hydrogen bonds to asparagine 71 and serine 272 is within

hydrogen bond distance to the pyridine ring nitrogen of PLP.28 Given the pKa difference

between serine 272 (~15) and the pyridine ring nitrogen (~5), one would not expect

protonation of the pyridine ring nitrogen.

OASS catalyzes the second stage of cysteine synthesis, converting O-acetyl-L-

serine (OAS) and bisulfide to L-cysteine via a ping-pong mechanism (Figure 1.7). In the

resting state the enzyme exists as a Schiff base between lysine 41 and C4' of the cofactor

(ε412 nm = 7,620 M-1 cm-1).29 Upon introduction of OAS the external aldimine is

formed by transimination. OASS, as with other PLP utilizing enzymes, holds the bond to

be cleaved orthogonal to the pyridine ring of PLP in accordance with Dunathan’s

hypothesis. An additional constraint is maintaining the leaving group, acetate, in such a

position that it is anti to the Cα proton, in accordance with an anti E2 elimination

pathway.28 Abstraction of the Cα proton, the largely rate determining step, is followed by

elimination of acetate and formation of a stable aminoacrylate, with maximal absorbance

at 330 nm and 470 nm (ε470 nm = 9760 M-1 cm-1).29 Maintaining the active site lysine in

a protonated state (pKa ~8) helps protect the aminoacrylate from abortive
 14

transimination.28 The second half reaction consists of attack by the bisulfide nucleophile

on the β-carbon of the aminoacrylate and protonation at Cα, thus forming the L-cysteine

external aldimine. Transimination with lysine 41 returns the enzyme to the resting state

with the release of L-cysteine. Spectral changes associated with formation of the stable

aminoacrylate intermediate (470 nm) from the internal aldimine (412 nm) allow for direct

monitoring of the rate determining first half reaction by stopped flow kinetics.

As with alanine racemase, no directly observable quinonoid is detected with the

wild type OASS enzyme. Mutation of serine 272 to aspartate failed to generate an

observable quinonoid intermediate28, whereas the corresponding mutations of arginine

219 in alanine racemase resulted in an observable quinonoid intermediate. It is

postulated that by employing a concerted mechanism for elimination (an E2 pathway)

OASS bypasses the quinonoid intermediate (Figure 18).28 This is thought to make OASS

unique among β-elimination enzymes: tryptophan synthase (fold type II), tryptophanase

(fold type I) and tyrosine phenol lyase (fold type I) are all thought to catalyze elimination

through a stepwise E1 mechanism involving quinonoid formation.28

 15

Figure 1.1. Vitamin B6 structures and numbering convention.

 16

Figure 1.2. Mechanistic themes common to PLP utilizing enzymes.

17
 18

Figure 1.3. Orientation of the Cα-H σ bond (labeled H) to be cleaved is held orthogonal to the

plane of the pyridine cofactor ring. Hyperconjugation with the π electron system selectively

weakens the Cα-H σ bond.

H
 19

Figure 1.4. Interaction of arginine 386 and 292 with the dicarboxylate groups of the

substrate analog α-methyl L-aspartate holds the α-methyl group orthogonal to the plane

of the cofactor ring.

 20

Figure 1.5. Aspartate aminotransferase mechanism.

21
 22

Figure 1.6. Alanine racemase mechanism.

 23

Figure 1.7. O-Acetylserine sulfhydrylase mechanism.

 24

Figure 1.8. Comparison of elimination by an E1 versus an E2 pathway. The E2 pathway

bypasses the formation of the quinonoid intermediate.30

 25

References

1. Gyorgy, P., Developments leading to the metabolic role of vitamin B6. The
American Journal of Clinical Nutrition 1971, 24, 1250-1256.

2. Gyorgy, P., The history of vitamin B6. The American Journal of Clinical Nutrition
1956, 4, 313-317.

3. Gyorgy, P., Vitamin B2 and pellagra-like dermatitus in rats. Nature 1934, 133,
498.

4. Snell, E. E., Metabolic significance of B-vitamins. Physiol. Rev. 1953, 33, 509-
524.

5. Wood, W. A., The discovery, synthesis, and role of pyridoxal phosphate: phase I
of many phases in the Gunsalus odyssey. Biochemical and Biophysical Research
Communications 2003, 312, 185-189.

6. Metzler, D. E. I., M.; Snell, E.E., A general mechanism for vitamin B6-catalyzed
reactions J Am Chem Soc 1954, 76, 648-652.

7. According to the Science Citation Index as of August, 2010

8. Eliot, A. C.; Kirsch, J. F., Pyridoxal phosphate enzymes: mechanistic, structural,

and evolutionary considerations. Annu Rev Biochem 2004, 73, 383-415.

9. Toney, M. D., Reaction specificity in pyridoxal phosphate enzymes. Arch

Biochem Biophys 2005, 433, (1), 279-87.

10. Bugg, T., An Introduction to Enzyme and Coenzyme Chemistry. Blackwell

Sciences Ltd: Oxford, 1997.

11. Garrett, R. H. G., C.M., Biochemistry. Brooks/Cole: Boston, 2009.

12. Braunstein, A. E., Pyridoxal Phosphate. Academic: New York, NY, 1960; Vol. 2.

13. Metzler, D. E., This Week's Citation Classic. Current Contents 1985, 16, 20.
 26

14. Percudani, R. P., A., A genomic overview of pyridoxal-phosphate-dependent

enzymes. EMBO Rep. 4 2003, 850-854.

15. Hayashi, H., Pyridoxal enzymes: mechanistic diversity and uniformity. J Biochem
1995, 118, (3), 463-73.

16. Dunathan, H. C., Conformation and reaction specificity in pyridoxal phosphate

enzymes. Proc Natl Acad Sci U S A 1966, 55, (4), 712-6.

17. Okamoto, A.; Higuchi, T.; Hirotsu, K.; Kuramitsu, S.; Kagamiyama, H., X-ray
crystallographic study of pyridoxal 5'-phosphate-type aspartate aminotransferases
from Escherichia coli in open and closed form. J Biochem 1994, 116, (1), 95-107.

18. Stamper, G. F.; Morollo, A. A.; Ringe, D., Reaction of alanine racemase with 1-
aminoethylphosphonic acid forms a stable external aldimine. Biochemistry 1999,
38, (20), 6714.

19. Kurokawa, Y.; Watanabe, A.; Yoshimura, T.; Esaki, N.; Soda, K., Transamination
as a side-reaction catalyzed by alanine racemase of Bacillus stearothermophilus. J
Biochem 1998, 124, (6), 1163-9.

20. Schneider, G.; Kack, H.; Lindqvist, Y., The manifold of vitamin B6 dependent
enzymes. Structure 2000, 8, (1), R1-6.

21. Hayashi, H.; Mizuguchi, H.; Miyahara, I.; Islam, M. M.; Ikushiro, H.; Nakajima,
Y.; Hirotsu, K.; Kagamiyama, H., Strain and catalysis in aspartate
aminotransferase. Biochim Biophys Acta 2003, 1647, (1-2), 103-9.

22. Hayashi, H.; Mizuguchi, H.; Kagamiyama, H., The imine-pyridine torsion of the
pyridoxal 5'-phosphate Schiff base of aspartate aminotransferase lowers its pKa in
the unliganded enzyme and is crucial for the successive increase in the pKa
during catalysis. Biochemistry 1998, 37, (43), 15076-85.

23. Meister, A.; Sober, H. A.; Peterson, E. A., Studies on the coenzyme activation of
glutamic-aspartic apotransaminase. J Biol Chem 1954, 206, (1), 89-100.

24. Shaw, J. P.; Petsko, G. A.; Ringe, D., Determination of the structure of alanine
racemase from Bacillus stearothermophilus at 1.9-A resolution. Biochemistry
1997, 36, (6), 1329-42.
 27

25. Sun, S.; Toney, M. D., Evidence for a two-base mechanism involving tyrosine-
265 from arginine-219 mutants of alanine racemase. Biochemistry 1999, 38, (13),
4058-65.

26. Inagaki, K.; Tanizawa, K.; Badet, B.; Walsh, C. T.; Tanaka, H.; Soda, K.,
Thermostable alanine racemase from Bacillus stearothermophilus: molecular
cloning of the gene, enzyme purification, and characterization. Biochemistry
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27. Spies, M. A.; Toney, M. D., Multiple hydrogen kinetic isotope effects for
enzymes catalyzing exchange with solvent: application to alanine racemase.
Biochemistry 2003, 42, (17), 5099-107.

28. Tai, C. H.; Cook, P. F., Pyridoxal 5'-phosphate-dependent alpha,beta-elimination

reactions: mechanism of O-acetylserine sulfhydrylase. Acc Chem Res 2001, 34,
(1), 49-59.

29. Tai, C. H.; Rabeh, W. M.; Guan, R.; Schnackerz, K. D.; Cook, P. F., Effect of
mutation of lysine-120, located at the entry to the active site of O-acetylserine
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30. Figure 1.8 was taken from reference 28.