Recent topics in pyridoxal 5'-phosphate enzyme studies.

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Annu. Rev. Biochem. 1990.59:87-110. Downloaded from www.annualreviews.org Access provided by State University of New York - Stony Brook on 12/22/14. For personal use only.

Annu. Rev. Biochem. 1990. 59:87-110 Copyright © 1990 by Annual Reviews Inc. All rights reserved

RECENT TOPICS IN PYRIDOXAL 5'-PHOSPHATE ENZYME STUDIES Hideyuki Hayashi Department of Medical Chemistry, Osaka Medical College, Takatsuki, Osaka 569, Japan

Hiroshi VVada Department of Pharmacology II, Osaka University School of Medicine, Nakanoshima, Osaka 530, Japan

Tohru Yoshimura, Nobuyoshi Esaki, and Kenji Soda Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan KEY WORDS:

aminotransferase, decarboxylase , racemase, pyridoxal 5' -phosphate enzymes.

CONTENTS INTRODUCTION.....................................................................................

88

AMINOTRANSFERASES........................................................................... Aspa rta te Am ino transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A roma tic Am ino A cid Am ino trans fe rase and Ty ros ine Am inotrans fe rase . . . . . . . . . . . . . . D-Am ino Ac id Am ino trans fe rase . . . . . . . . . . . . . . ... . .................................. Orn ith ine 8-A m ino trans fe rase . .. . . . ...... . ..... ... . ........... . ... . ............. . . ....

88 96 97 99

DECARBOXYLASES................................................................................ A roma tic Am ino A cid De ca rbo x ylase ..... . . . .... . . . . . . ... . . . . ...... . . . . . . . . . . . . ... ... . . . . .... H is tid ine De ca rbo xylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gl utama te De ca rbo x ylase ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100 100 101 102

RACEMASES.......................................................................................... A l an ine Race ma se . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a-Am ino-e··Cap rola ctam (A CL) Ra cemase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

102 102 105

CONCLUDING REMARKS........................................................................

105

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88

87

0066-4154/9010701-0087 $02.00

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HA Y ASHI ET AL


INTRODUCTION Pyridoxal 5' -phosphate (PLP) catalyzes versatile reactions in either enzyme bound or free form: transamination, decarboxylation, racemization, elimina tion, and so on. These reactions are facilitated by the electron withdrawing nature of the pyridine moiety, which labilizes the bonds around the Co: atom through the extended 7T-system of the Schiff bases formed with amino acids. The rates of these reactions are tremendously enhanced by the functional groups of the protein portions of PLP enzymes. Moreover, the protein de termines the reaction specificity; it reinforces one of the reactions and ex cludes the others that are inherently catalyzed by PLP. Therefore, how the protein portions of PLP enzymes participate in catalysis has been of great interest. Various PLP enzymes have been studied at the molecular level by means of X-ray crystallography and genetic techniques as summarized in Table 1. We describe here some current developments on selected PLP enzymes whose study has progressed sufficiently to give a clue to understand ing how the protein functions in selective catalysis. AMINOTRANSFERASES

Aspartate Aminotransferase Aspartate aminotransferase (EC 2.6.1.1) (AspAT), which was formerly called glutamic oxalacetic transaminase (GOT), has been studied most ex tensively and is representative of PLP enzymes (74). It is a dimer of identical subunits with a molecular weight of about 45,000, and contains one PLP molecule per subunit. It is widely distributed among animals, plants, and m icroorganisms. In eukaryotes, the enzyme exists as isoenzymes in cyto plasm and mitochondria. The sequence homology between these isoenzymes is close to 45% in all species studied. Apparently, the isoenzymes have evolved from a common ancestral AspAT by gene duplication. AspAT was isolated in pure form from pig heart by Jenkins et al (75) in 1959; the preparation was later found to be cytosolic AspAT. Since then, . extensive investigations have been carried out on both the cytosolic and mitochondrial (76) enzymes. Today, the mechanisms of action of both isoen zymes (74) and of the E. coli enzyme (77) are being investigated at the molecular level based on their X-ray structures. Characterization of AspAT, through kinetic, physiological, crystallographic, as well as biological and clinical investigations, is thoroughly reviewed in (74). More up-to-date in formation is provided by the excellent review of Jansonius & Vincent (78). Recent advances in genetic engineering have marked an era in the study of PLP-enzymes, in particular AspAT. They made possible the sequencing of a variety of enzymes from various sources and in vitro mutagenesis of a specific residue involved in the function of the enzyme.


Table 1

PLP-dependent enzymes whose primary structure has been determined Lysine residue

Total amino Enzyme

EC number

serine hydroxymethyltransferase

2.1.2.1

aspartate AT

2.6.1.1

Source

acid residues

Mr

Refs.

Subunits

Gene

binding PLP

glyA

256K

1

229K

2

246K

3-5

256K

6

Escherichia coli

417

45,316

4

rabbit

483

52,844

4

E. coli

396

43,573

2

chicken cytoplasm

410

45,659

2

aspC

7,8 (cryst) 9

horse cytoplasm

412

46,213

2

258K

mouse cytoplasm

412

46,100

2

258K

to

pig heart muscle

412

46,344

2

258K

ll, 12

chicken mitochondria

401

44,933

2

250K

13

horse mitochondria

401

44,567

2

250K

9

cytoplasm

(precursor)

human mitochondria

401

44,693

2

250K

14

mouse mitochondria

430

47,411

2

279K

10

pig mitochondria

401

44,665

2

250K

15, 16

rat mitochondria

401

44,249

2

250K

17, 18

2

tyrosine AT

2.6.1.5

rat

454

50,634

alanine-valine AT

2.6.-.-

E. coli

226

24,753

19 avtA

20

hisC

22

hisS

23

succinyldiaminopimelate AT

2.6.1.17

E. coli

274

30,039

histidinolphosphate AT

2.6.1.9

E. coli

356

39,316

Saccharomyces cerevisiae

384

42,541

branched-chain amino acid AT

2.6.1.42

E. coli

309

34,093

6

ilvE

Salmonella typhimurium

308

33,920

6

ilvE

159K

25

human

439

48,534

oat

292K

26

rat

439

48,332

292K

27

S. cerevisiae

423

46,042

ornithine AT

2.6.1.13

21

4 car2

24

28


Table 1

(Continued) Total amino Source

acid residues

Lysine residue

Enzyme

EC number

aromatic amino acid AT

2.6.1 .

E. coli

397

43,537

D-amino acid AT

2.6 . 1 .21

Bacillus sp. YM-J

282

32,226

aromatic-L-amino acid DC

4 . 1 . 1 .28

Drosophila melanogaster

511

56,771

503

(isozyme

M,

Subunits

Gene

binding PLP

Refs.

247K

4

145K

29

dde

338K

30

56,346

ddc

330K

30

tyrB

2

a)

D. melanogaster (isozyme I) diamino-pimelate DC

4.1.1.20

E. coli

420

46,177

lysA

31

histidine DC

4.1 . 1 .22

Morganella morganii

377

42,744

hde

32

ornithine DC

4. 1 . 1 . 1 7

mouse

46 1

5 1 , 1 63

ode

rat

46 1

5 1 ,470

36

tryptophanase

4. 1 .99.1

33-35

Trypanosoma brucei

445

49,1 76

37

S. cerevisiae

466

52,285

38

E. coli

471

5 2,674

442

47,900

dsdA

1 l8K

514

56,195

ilvA

62K 58K

D-serine dehydratase

4.2.1 . 1 4

E. coli

threonine dehydratase

4.2. 1 . 1 6

E. coli (biosynthetic)

4

39,40

tnaA

4 1 ,42 43

E. coli

tryptophan synthase

44

(catabolic)

329

35,232

4

tde

S. cerevisiae

576

63,8 1 8

4

ilvl

45

4 . 2 . 1 .20

S. cerevisiae

707

76,625

a-chain

Bacillus subtilis

267

29,450

Brevibaeterium

28 1

29,222

lactofermentum

2

a2 {32 a2{32

trp5

46

trpA

47

trpA

48


f3-chain

E. coli

268

28,724

a2f32

Enterobactor aerogenes

269

28,557

a2f32

Pseudomonas aeruginosa

267

28,417

a2f32

S. typhimurium

268

28,670

a2f32

B. subtilis

400

43,691

a2,B2

trpA

49-51

trpA

54

52,53 50,54

trpB

90K

47

Br. lactoJermentum

416

44,704

a2f32

trpB

99K

48

E. coli

396

42,851

a2f32

trpB

86K

49

Ps. aeruginosa

402

43,607

a2f32

trpB

93K

54

S. typhimurium

396

42,805

a2f32

trpB

86K

55

386

41,550

4

metB

198K

56

thrC thrC

59K

57

\o7K

58

metC

210K

59

41K

60

39K

61 62

cystathionine y-synthase

4 .2.99.9

E. coli

threonine synthase

4.2.99.2

B. subtilis

352

37,463

E. coli

428

47,1l3

395

43,212

cystathionine {3-1yase

4.4.1.8

E. coli

alanine racemase

5.1.1.1

B. subtilis

389

43,290

B. stearothermophilus

386

43,341

359

39,075

air

34K

dadB

35K

4

2

S. typhimurium (biosynthetic)

S. typhimurium (catabolic precursor) a-amino €-caprolactam racemase

5.1.1.

glycogen phosphorylase

2.4.1.1

63

356

38,804

435

45,568

human

847

97,221

4

681K

65

rabbit

842

97,183

4

680K

66

Acromobacter obae

64

67, 68 (cryst.)

S. cerevisiae

891

102,439

740K

69

a-glucan phosphory lase

2.4.1.1

potato

916

\03,654

762K

70

maltodextrin phosphorylase

2.4.1.1

E. coli

797

90,405

646K

71

2.7.7.27

E. coli

431

48,740

g/gC

39K, 195K

72

S. typhimurium

431

48,583

glgC

39K, 195K

73

glucose

I-phosphate

transferase

adenylyl-


92

HAYASHI ET AL

Mitochondrial AspAT (mAspAT) is synthesized in the cytoplasm on free ribosomes and is transported into the mitochondria by a specific processing mechanism (74). The primary structure of the precursor form of pig mAspAT (premAspAT) was deduced by sequencing the cDNA clone (79). The pre cursor has an extra group of 29 amino acid residues (the "presequence") at the N-terminal. The presequence of mAspAT showed no significant homology with those of other mitochondrial enzymes, but they have in common the presence of several basic amino acid residues and the absence of acidic amino acid residues (6). Later, the cDNA clones for chicken (80), mouse (0), and rat (8 1) premAspAT were obtained. Expression of the cDNA for premAspAT in E. coli was unsuccessful, owing to the tendency of premAspAT to aggre gate. In contrast, the cDNA for the mature form of mAspAT was expressed and the product was soluble and catalytically fully active (82). An in vitro transcription and translation system for the cDNA of pig premAspAT was constructed by inserting the cDNA into pSP65. PremAspATs with alterna tions within the presequence were then synthesized and analyzed for the rate of both import and processing (83). The results showed that the N-terminal 8 residues are essential for both import and processing, while the Arg at position 28 (second from the last) of the presequence may be the recognition site for the processing enzyme in the mitochondria. The import of pre mAspAT into mitochondria is dependent on the energy-generating system of mitochondria, and thus is inhibited by an uncoupling reagent (74). Pre mAspAT, accumulated in the cytoplasm, is degraded by cytosolic proteolytic 2 enzymes dependent on ATP and Mg +, with a half-life of about 5 min (84). Recently, rat premAspAT was expressed in E. coli using the pKK223-3 vector (85). The isolated precursor exists as a soluble dimeric protein, con tains one PLP per subunit, and shows full catalytic activity . Clearly, the presequence peptide does not affect the structural and functional integrity of mAspA T. This fact will facilitate the elucidation of the mitochondrial import mechanism of mAspAT at the molecular level. cDNAs for cAspAT of pig (86) , mouse ( 10), and rat (87) were obtained. cDNA for rat cAspAT was expressed in E. coli as a hybrid protein with the N-terminal 1 1 residues of f3-galactosidase (87). cDNA for pig cAspAT was directly expressed in E. coli by insertion into pKK223-3 (86), and the expressed enzyme was about 3% of the total cellular soluble protein. The recombinant pig cAspAT showed heterogeneity in the amino-terminal se quence, with deletion of 1-3 residues at the N-terminal end. However, these recombinant cAspATs are spectrophotometrically and kinetically in distinguishable from the native enzymes. Thus small additions or deletions of residues in the N-terminal region did not affect the catalytic activity and structural integrity of cAspAT. The genome structures showed that there are high levels of homology


PYRIDOXAL 5'-PHOSPHATE ENZYMES

93

between the promoter regions of cAspAT and mAspAT, cAspAT and cytosol ic malate dehydrogenase, and mAspAT and mitochondrial malate de hydrogenase (88-90). They lack the characteristic TATA and CAAT boxes, contain G+C-rich sequences, as well as the sequences compatible with the formation of stable stem-loop structures, and have multiple transcription initiation sites. These four enzymes participate in the malate-aspartate shuttle (91), and the sequence homologies indicate the possibility that these isoen zymes are expressed in a coordinated fashion. The sequence of the coding frame shows that 5 of the 8 introns in the cAspAT gene and 9 introns in the mAspAT gene are at identical positions (88, 89). This indicates that at least in AspAT, the introns already existed before the divergence of cAspAT and mAspA T, and suggests that certain introns are phylogenetically very old and may have been possessed by ancestral prokaryotes. E. coli AspAT has kinetic and structural properties almost identical with those of the mammalian enzymes (92, 93). It can, however, transaminate several aromatic amino acids (92, 94). Similarly, aromatic amino acid ami notransferase (EC 2.6. 1.57) of E. coli has significant activity for transaminat ing Asp with a-ketoglutarate (95, 96); these two enzymes are structurally similar (95). AspAT is encoded by the aspC gene in E. coli (97), and the nucleotide sequence has been elucidated (98). The AspAT was overproduced several hundredfold in E. coli by insertion of its structural gene into a high copy plasmid, pUC19 (99). The expression system thus established is used for site-directed mutagenesis of catalytically important residues. Malcolm & Kirsch ( 100) were the first to report the construction of mutant AspAT. They observed that substitution of Ala for Lys258 (K258A), which is the PLP binding lysine, completely abolished activity of the enzyme. Kuramitsu et al found that the mutant enzyme with Lys258 replaced by Arg showed 2-3% activity compared to the wild-type enzyme (101). These results are in accor dance with the view that Lys258 acts as an acid/base catalyst for the 1,3prototropic shift in aldimine/ketimine interconversion. This idea was verified later by the finding that catalytic activity of the inactive enzyme (K258A) is restored by exogenous amines, which will functionally replace the deleted catalytic group (102). X-ray crystallography suggested that Tyr70 may also be involved in the proton transfer reaction because of its proximity to coenzyme moiety. This possibility was, however, discounted by the observation that a mutant with Tyr70 replaced by Phe retained 15% of the maximal activity of the wild-type enzyme (103). Tyr70 seems to be involved in the binding of the coenzyme phosphate group, for the mutant enzyme loses the coenzyme faster than the wild-type enzyme. In any case, the presence of an acid/base group on the si side of the external aldimine or ketimine assures that ster eospecific protonation occurs from the si side. Protonation from the re side to form the external aldimine of PLP with o-glutamate, probably medi-


94

HAYASHI ET AL

ated by a water molecule, takes place only once in 1.5 x 107 transamination cycles of glutamate with a-ketoglutarate (lOS). Protonation to form the ketimine of pyridoxamine 5' -phosphate (PMP) with a-ketoglutarate also occurs from the si side ( 1 06). Apparently, in the reverse reaction, the pro-S H atom of C4' of the PMP moiety of ketimine should be labilized. Interestingly, labilization of the pro-S H of PMP does not require the formation of ketimine; the pro-S 3H of PMP exchanges with solvent water when apoAspAT is reconstituted with eH]-PMP ( 1 07), and the rate was enhanced 400-fold in the presence of amino acid substrates (108). Recent studies by Kirsch's group provide interesting information about the transition state structure ( l 09). In the reaction of pig cAspAT with aspartate, the 2H20 solvent kinetic isotope effect was independent of the presence of a-2H, and conversely, the kinetic isotope effect of a_2H is unaffected by 2H20 solvent. These results indicate that both isotopic substitutions involve protons in a single transition state, and thus the 1 ,3-prototropic shift occurs in a concerted fashion . This mechanism requires the close association of Lys258 with both a-H of the substrate and C4' of the coenzyme. On the other hand, the isotope effect on the reaction of pig rnAspAT with glutamate shows that a-proton abstraction is largely rate determining, suggesting a two-step mech anism for the 1,3-prototropic shift. This fact is in agreement with the finding that a small fraction of the a-proton of glutamate is transferred to the C4'-position of PMP in the reaction catalyzed by chicken cAspAT ( 1 06). These observations suggest that at least in pig cAspAT, the amino group of Lys258 is oriented to be in close contact with the external aldimine. The protonated aldimine has an absorption band at 430 nm (74), which is usually accompanied by a minor absorption band at 330 nm. The latter band is ascribed to the enolimine tautomer of the protonated aldimine. In the former ketoenamine tautomer, the extended 7T-orbital and the (T-orbital of Ca-H are separated by >NH, and the electron withdrawing action of the pyridine nitrogen is interrupted at this position. Thus the ketoenamine tautomer is unfavorable for catalysis. Resolution of the spectra with lognormal curves ( 110) and 19F NMR study by Metzler's group ( 111) of apoAspAT reconsti tuted with a coenzyme analogue, 6-F-PLP ( 1 1 1), suggested a role for tautomerism from ketoenamine to enolimine in the reaction catalyzed by AspAT. ApoAspAT reconstituted with 3-0-methyl-PLP, which will form only the enolimine tautomer, reacted with the amino acid substrate to form a stable quinonoid structure, which is consistent with the above notion ( 1 12). Resonance Raman spectra were measured on a PLP form of pig cAspAT (74, 113). The assignments of resonance peaks to specific vibrational modes were based on a model compound, the Schiff base of PLP with valine (114). The results showed the protonation of both pyridine N atom and imine N atom in the native enzyme at pH 5.0, and in the enzyme-inhibitor complex at pH 8.5.



95

The only discrepancy was that the C=N (imine) stretching frequency of cAspAT at pD 5.0 had an unusually low value, owing to a novel HID exchange reaction at the C4 I atom of PLP in cAspAT caused by the laser irradiation at 406.7 nm used in the resonance Raman spectra measurement (113). This unique photochemical HID exchange reaction observed in cAsp AT must be ascribed to the polarization of the phenolate 0 atom by Tyr225 via a hydrogen bond, which prevents the electrons of the phenolate 0 from flowing into the imine N atom; thus a p-quinoid structure rather than an o-quinoid structure evolves upon photoinduction of cAspAT (1 1 3 ). In the normal enzymatic catalysis, the p-quinoid structure of the PLP-substrate complex in cAspAT seems to be stabilized by polarization of the phenolate 0 atom by Tyr225 ( 1 13). Site-directed mutations were also performed on substrate-binding residues (Arg292 and Arg386). Replacement of Arg292 by Asp ( 1 15), and by Val or Leu ( 116), enhanced the activity toward basic and aromatic amino acids, respectively, with nearly complete loss of activity with dicarboxylic amino acids. This result is consistent with the X-ray crystaUographic data that Arg292 forms the binding site for the side chain of the dicarboxylic substrate. Arg386, on the other hand, is the binding site for the a-carboxylate group of the substrate. Substitution of Lys for Arg386 resulted in a decrease in kcatlKm values by a factor of 1 04, i.e. an increase in activation free energy of about 5 kcallmol for dicarboxylic substrates (117). Substitution of Lys for Arg292 produced a similar change in kcatfKm values (1 17). This value is close to that caused by [Arg17 1 Lys] mutation of Bacillus stearothermophilus lactate de hydrogenase (1l 8), indicating the large contribution of the complementary hydrogen bond formation between Arg and a carboxylate group in the transi tion state and the preferential occurrence of Arg as the binding site for the carboxylate group. However, the resolution of kcatlKm values to kcat and Km revealed that the mutation in AspAT caused a 103-fold decrease in kcat values, whereas the same mutation in lactate dehydrogenase did not change the kcat value. Clearly this shows that the Arg involved in substrate binding also participates in the catalytic reaction in AspAT, but not in lactate de hydrogenase. These findings should be related to the observation that ligand binding causes conformational changes involving the "small" domain move ment and coenzyme "tilt," which are essential for efficient catalysis (74). Further studies on AspAT, from various points of view, are now under way in many laboratories. In the next 10 years, a vast amount of new information will be accumulated, and the mechanism will be revealed at the atomic level. AspA T activity in serum has long been used, together with that of alanine aminotransferase, for the diagnosis of hepatitis and cardiac infarction (74). In the acute phase of experimental liver ischemia, cytosolic enzymes, such as alkaline phosphatase ( 1 1 9), leak from liver. cAspAT is, however, inactivated


96

HAYASHI ET AL

rapidly by the simultaneously released lysosomal enzymes, and does not appear in serum (120). On the other hand, mAspAT is protected by the mitochondrial membrane from the attack of lysosomal enzymes. The leakage of mAspAT from the matrix does not occur if the ischemia is reversible; the complete loss of phosphorylating activity of mitochondria and the consequent disorganization of cell structure is needed for mAspAT to appear in serum (120). Thus the cumulative activity of mAspAT, not cAspAT, correlates well with the extent of liver necrosis. mAspAT may also be used as a good marker of chronic, but not of acute alcoholism (121). Aromatic Amino Acid Aminotransferase and Tyrosine Aminotransferase Aromatic amino acid aminotransferase (EC 2.6.1.57) (AroAT) is widely d istributed in microorganisms (122), and catalyzes the last step of the biosynthetic route for phenylalanine and tyrosine. In E. coli, "transaminase A" was reported to catalyze the transamination between aspartate and aromat ic amino acids with a-ketoglutarate ( 123). Subsequently transaminase A was divided into two separate enzymes (124): AspAT, encoded by the aspC gene, and an aromatic amino acid aminotransferase with high activity toward both aromatic amino acids and aspartate, which is encoded by the tyrB gene and is repressed by tyrosine (125, 126). The nucleotide sequence of tyrB has been determined (127), and showed that AroAT and E. coli AspAT are homologous proteins with an amino acid sequence homology of 44%. The tyrB gene inserted into pUC 19 is highly expressed in E. coli (128). The physicochemical properties of the overproduced enzyme was investigated, and it was observed that the properties of E. coli AspAT and AroAT are quite similar, as is anticipated from their high homology (128). Crystals of AroAT large enough for X-ray analysis have not been obtained so far. Therefore, the three-dimensional structure is unclear at present. Model-building studies using the backbone fold of chicken mitochondrial AspAT as the initial template for AroAT were attempted (129). The results showed that only minor changes are required to accommodate the substituted side chains, and suggest that the structural motif of AroAT is quite similar to that of AspAT. Tyrosine aminotransferase (EC 2.6. 1.5) (TyrAT) is a liver-specific enzyme catalyzing the first reaction of the degradation pathway of Tyr to fumarate and acetoacetate, and is the rate-limiting enzyme for Tyr catabolism (74). Gluco corticoids, cAMP, and a high protein diet all increase the activity, whereas administration of glucose decreases it (74). Both cAMP and glucocorticoid cause several hundredfold increases in TyrAT mRNA (130). Presumptive sequences were proposed for the glucocorticoid-receptor complex binding site (131) and for the binding site for a cAMP-CAP-like protein complex (132) in the 5f-end region of the TyrAT gene of rats.


97


Study of the primary structure of rat TyrAT (133, 134) showed that the invariant, functional residues of AspAT and AroAT are conserved in TyrAT, although the sequence homologies bctween TyrAT and pig cAspAT are only 8% and 15%, respectively. These results show that the folding of the polypeptide chain of TyrA T is similar to those of AspAT and AroAT, and suggest that these enzymes belong to a superfamily. D-Amino Acid Aminotransferase In cooperation with amino acid racemases, o-amino acid aminotransferase (Ee 2.6. l .21) (o-AAT) plays an essential role in the biosynthesis of o-amino acids, which are used as constituents of the peptidoglycan layer of bacterial cell walls. o-AATs have been found in bacteria of the genus Bacillus, Rhodospirillum rubrum, Rhizobium japonicum, and also germinating pea seedings (74). The enzyme was purified to near homogeneity from B. subtilis (135-137), and B. sphaelicus (138-140). Recently, thermostable o-AAT has been purified from a newly isolated thermophilic bacteria, Bacillus species YM-l (141), and its gene has been cloned and expressed efficiently in E. coli (24). It has a molecular weight of about 62,000 (141) and consists of two subunits identical in molecular weight (30,000). The native holoenzyme exhibits absorption maxima at 279, 333, and 415 nm at pH 7.4 and contains one mole of PLP per subunit. The addition of o-alanine to the enzyme solution causes a decrease in absorbance at 419 nm and an increase at 335 nm, suggesting the conversion of the PLP form of the enzyme into the PMP form. Treatment of the enzyme with phenylhydrazine yields a semiapoenzyme, which is catalytically inactive in the absence of added PLP, and which no longer exhibited an absorption maximum at 415 nm. Semiapoenzyme still has the peak at 333 nm, which is due to the bound cofactor and can be detected as PLP (about 0.2 mol per mol of subunit). The circular dichroism spectrum of the thermostable enzyme shows a positive peak at 280 nm and negative peaks at 333 and 418 nm. These properties of thermostable o-AAT are similar to those observed in the enzyme from B. sphaelicus (138-140). The substrate specificity of the Bacillus YM -1 enzyme is similar to that of the B. subtilis enzyme, which reacts more specifically with o-alanine, o-glutamate, o-aspartate, o-asparagine, and o-a-aminobutyrate than the B. sphaelicus enzyme. The primary structure of thermostable o-AAT has been determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product. The polypeptide composed of 282 amino residues is as follows: IGYTLWNDQIV KDEEVKIDKE 21DRGYQFGDGV YQVVKVYNGE 41MFTVNEHIDR LYASAEKIRI 61TIPYTKDKFH QLLHELVEKN

98

HA YASHI ET AL 81ELNTGHIYFQ VTRGTSPRAH IOIQFPENTVKPV IlGYTKENPR l2lPLENLEKGVK ATPVEDIRWL 141 RCDIKSLNLL GAVLAKQEAH 161 EKGCYEAILH RNNTVTEGSS 181SNVFGlKDGl LYTHPANNMI


201LKGITRDVVI ACANEINMPV 221KEIPFTTHEA LKMDELFVTS 2 41TTSEITPVIE IDGKLIRDGK 261VGEWTRKLQK QFETKIPKPL

The calculated molecular weight is 32 ,226, and the cofactor PLP is bound to Lys 1 45. Based on gene analysis, the correlation between structure and function of D-AAT has been studied by site-directed mutagenesis. o-AAT contains eight cysteine residues. Since the titration of four cysteine residues in the B. sphae/icus enzyme with 5,5' -dithiobis(2-nitrobenzoic acid) (DTNB) led to loss of activity (139, 142), one or more of the SH groups may have a catalytic role. To clarify this role, each of the three cysteinyl residues per subunit in the thermostable o-AAT was changed to a glycine residue (143). Two of the three Cys mutants (C 164G and C142G) were as active as the wild-type, and the third mutant (C2 1 2G) retained 90% of the activity of wild-type enzyme. Therefore, it is clear that SH groups of the enzyme are not essential for its catalytic activity. Conversion of Ser 146, adjacent to Lys 145 to which PLP is bound, to an alanine residue does not alter the catalytic activity but has a significant effect on the SH titration behavior with DTNB (143). With the wild-type enzyme, only one SH per dimer is titrated after 4 h-exposure to 1 mM DTNB; however, under the same conditions, three to four Cys residues are titrated in the Ser mutant (S146A) with a concomitant loss in activity. In the presence of substrate D-alanine, the titration behavior of the S146A mutant is similar to that of the wild-type enzyme. This result suggests that the Cys residue(s) which is titrated in the S146A mutant could be at or near the active site, and the presence of a thionitrobenzoate moiety at (or near) the active site disturbs the enzyme. The accessibility of the SH group in the wild-type enzyme from the thermostable Bacillus is much lower than that for the enzyme from the mesophile source. Perhaps this themlOstable enzyme adopts a more compact structure, and Ser146 is important for this structure. Site-directed mutagenesis has been employed also to study the role of Trp139, which is close to the active site Lys145 (144). This tryptophan residue has been changed to Phe, Pro, Ala, His, and Asp. Only the mutant protein that has Phe substituted for Trp (W139F) is reasonably active (63% as active as the wild type) and stable. Fluorescence emission studies indicate that there is an energy transfer between Trp and PLP in the wild-type enzyme,


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which is almost absent in the W139F mutant. Fluorescence polarization studies (74) have revealed that in the PMP form of the enzyme, the rotational freedom of the cofactor is higher in the W139F mutant enzyme than in the wild type. These results suggest the importance of an aromatic side chain at position 139 in D-AAT and the influence of this residue on the behavior of the coenzyme PLP. The three-dimensional structure of the enzyme is being studied (145). The availability of large amounts of pure enzyme achieved by molecular cloning of the structural gene leads to interesting findings. A recent study has demonstrated that D-serine,which is a relatively poor substrate compared with o-alanine, serves as a unique type of inhibitor for o-amino acid aminotrans ferase (146). Incubation of D-serine with enzyme rapidly generates an absor bance band at 493 nm. This 493 nm band, which is considered to represent the quinonoid intermediate, is also observed but with a short half-life during the transamination of o-alanine. In the reaction with o-serine, the quinonoid intennediate is extremely stable,permitting it to accumulate. After accumula tion reaches a maximum, it slowly disappears concomitant with irreversible inactivation of the enzyme. During the reaction, o-serine undergoes turnover to yield a compound, which is believed to be {3-hydroxypyruvate. D-Alanine protects the enzyme from inactivation by o-serine. These results suggest that o-serine acts as an enzyme-activated inhibitor (suicide substrate) of D-amino acid aminotransferase. As o-AAT is the only transaminase that acts on a o-enantiomer of an amino acid, its evolution presents an interesting problem. A computer-aided search of the NBRF protein sequence data bank revealed that the branched chain L-amino acid aminotransferase of E. coli (24) has a significant sequence homology to the thennostable o-AAT (29). If the mutationally allowed substitutions for similar residues are taken into consideration, the similarity score increases to as high as 50,58,and 43% for the whole sequence,and the C- and N-terminal portions,respectively,suggesting that the structural genes for both enzymes may have evolved from a common ancestral gene.

Ornithine i)-Aminotransferase Ornithine 5-aminotransferase (BC 2.6.1.13) (OrnAT) catalyzes the second step of arginine degradation. The reaction product, glutamic semialdehyde, is converted either to glutamate or proline (74). OrnAT is a mitochondrial enzyme, and is synthesized in cytosol as a precursor polypeptide. The amino acid sequence of rat (27) and human (26) liver OrnAT precursor deduced from cDNA sequences showed 64% homology of the presequence and 91% homol ogy of the entire sequence. Residues 21-33, which contain the Ala25-Thr26 bond at which the lead peptide of rat OmAT is cleaved off (147),are identical in the two enzymes. The presequence of rat OrnAT shows 54% homology


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HAYASHI ET AL

with that of rat ornithine transcarbamoylase (148), while the sequence around Lys292, which binds PLP, closely resembles that in AspAT (148). Whether the catalytically important residues of AspAT are conserved or not is not clear, for the current structural analysis of OrnAT does not provide any information about the protein folding (149). However, several sequences can be compared to those of AspAT that contain the catalytic residues, i.e. Asp242-Pro243-Gly244-Tyr255 is related to Asp222-X-X-Tyr225, and Trp178-Gly179 is related to Trp140-Gly141 of mAspAT. Therefore, it is highly probable that the folding of OrnAT is similar to that of AspAT. OrnAT occurs also in various microorganisms (150), and has been crystal lized from B. sphaericus (151) and Corynebacterium sepedonicum (152). The enzyme is inducibly formed by the addition of L-Orn (150). The Bacillus enzyme catalyzes the stereospecific exchange of the pro-S hydrogen at the prochiral C5 carbon of L-Orn with the solvent (153). Both mammalian (154) and Bacillus enzymes (155) are irreversibly inactivated by gabaculine in a suicide fashion. L-Orn can be determined specifically by means of the Bacil lus enzyme (155). DECARBOXYLASES

Aromatic Amino Acid Decarboxylase Aromatic amino acid decarboxylase (EC 4.1.1.28) (AroDC) is a relatively nonspecific enzyme catalyzing decarboxylation of phenylalanine, tryptophan, and their hydroxylated derivatives (156), and is important for the formation of biogenic amines. AroDC purified from rat liver (157, 158) or pig kidney (159, 160) shows absorption maxima at 330 nm and 420 nm, characteristic of PLP-enzymes. The intensity ratios of the two peaks, E33o!E420, is 2.0-2.7 and considerably larger than that (about 0.5) of Morganella histidine de carboxylase, another PLP-dependent decarboxylase (168). Treatment of the enzyme with hydroxylamine abolished the absorbance at 420 nm and released PLP as an oxime. The resultant apodecarboxylase completely lost activity, which was restored by the addition of PLP. However, the apoenzyme had an absorption peak at 330 nm similar to that of the holoenzyme. The 330 nm-absorbing species attached to the enzyme could not be removed by 0.1 M NaOH. These properties are reminiscent of covalently bound pyrroloquino linequinone (PQQ); Duine's group showed that pig kidney enzyme contains I PLP and 1 covalently attached PQQ per dimeric enzyme molecule (161). The role of PQQ in AroDC, as that in GluDC, is not clear at present. Removal of PLP from AroDC completely inactivated the enzyme, but the activity was restored by supplementing with PLP (157, 158). This finding indicates that PLP is necessary for activity, but does not exclude the possibility that PQQ is also involved in the decarboxylation reaction. Duine & Jongejan proposed


101

that dopa is decarboxylated by fonning a Schiff base with PQQ, and the amine product is then transferred to PLP after which hydrolysis occurs (162).


Histidine Decarboxylase Like the other aromatic amino acids, histidine is decarboxylated by AA-DC (4.1.1.28) to some extent (156). There is, however, a distinct histidine decarboxylase (EC 4.1.1.22) (HisDC) specific for histidine (164), which is important for the formation of histamine. Despite the physiological importance of histamine, mammalian HisDC has not been fully characterized because of its limited amount in mammalian tissues and its instability (165, 166). HisDC was purified to homogeneity from fetal rat liver, which is thought at present to be the richest source of the enzyme (165, 166). An antibody raised against this preparation was used for immunohistological analysis. The results indicated that the distribution of HisDC correlates well with that of histamine, that is, HisDC is the specific decarboxylase for histidine. Moreover, it was found that HisDC is localized to a certain region of the brain, mainly the posterior hypothalamic area (166, 167). This is indirect but strong evidence for the presence of a histaminergic neuron system in brain. Apart from the difference in catalytic efficiency, Morganella HisDC and rat HisDC have similar properties, distinguished from those of pyruvoyl dependent HisDC (168, 169). a-Methylhistidine is a good competitive in hibitor of both enzymes, while histamine inhibits neither. 2-Mercaptoethanol is a mixed··type inhibitor. 3-N-Methylhistidine is a fairly good substrate, but I-N-methylhistidine is not. Both enzymes are inactivated by incubation with a-fluoromethylhistidine (168, 170). Extensive characterization of HisDC was carried out on the enzyme from Morganella AM-IS (168). The gene encoding HisDC has been cloned and sequenced (32). The sequence around PLP-binding lysine (Lys232) showed the conserved sequence Ser-X-His-(P-Pxy)-Lys earlier found in E. coli de carboxylases (32). The carbon and nitrogen isotope effects showed that the overall decarboxylation rate is jointly limited by the rate of transamination and by the intrinsic rate of decarboxylation (171), as is the case for glutamate decarboxylase. The carbon isotope effect of 1.05-1.06 on the decarboxylation step of HisDC is identical to that of GluDC (172), suggesting a similar transition state for the two enzymes. The value of a deuterium isotope effect for a-2H-histidine on the decarboxylation step was around 1.20, which is close to the value of the equilibrium isotope effect (1.29) for a deuterium attached to a carbon that changes hybridization from Sp3 to Sp2 (171). This suggests that the transition-state structure is close to Sp2, that is, the carboxyl group is almost broken in the transition state.

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Glutamate Decarboxylase Glutamate decarboxylase (EC 4.1.1. 15) (GluDC) has been the best studied enzyme among PLP-dependent amino acid decarboxylases. The enzyme from E. coli has been crystallized, and preliminary X-ray crystallographic data have been obtained (173). E. coli GluDC was proved to be a "pyridoxo-quinoprotein," which contains six PLP and six covalently attached PQQ per hexameric enzyme molecule (174). As PQQ does not occur free in E. coli, this result may suggest that covalently bound PQQ is synthesized in situ, namely in the quinoprotein itself (174).

RACEMASES

Alanine Racemase o-Alanine is a key building block of peptidoglycan, and is produced by alanine racemase (EC 5.1.1.1) from the L-isomer. The enzyme has been demonstrated in various bacterial strains and purified from Pseudomonas putida (175), Bacillus subtilis (176), Salmonella typhimurium (63, 177), Streptococcus faecalis (178), and Bacillus stearothermophilus (179).

Walsh and his coworkers have undertaken genetic and cloning approaches to identify alanine racemase genes (180). By using an E. coli strain that is temperature sensitive for growth in the absence of o-alanine, two distinct Salmonella DNA clones that complement the o-alanine requirement of the E. coli strain have been isolated (180). Both contain distinct alanine racemase genes, and each gene has been mapped, overproduced by means of strong promoters, and sequenced (62, 63, 177). One has been mapped at minute 37 on the chromosome and is termed the dadB alanine racemase gene, while the other has been mapped at minute 91 is termed the air gene (181). There are no additional alanine racemase genes detectable in Salmonella. Two distinct alanine racemase genes have been also demonstrated in E. coli ( l S2). The DadB enzyme is inducibly formed and functions in the catabolism of L alanine; o-alanine produced by racemization is deaminated to pyruvate by o-alanine dehydrogenase whose gene (dadA) is located adjacent to dadB and repressed together with dadB by dadR (ISO). The bacterium finally acquires two electrons by means of pyruvate oxidase, which oxidatively de carboxylates pyruvate to produce acetate. The other alanine racemase, AIr enzyme, is synthesized constitutively and functions in anabolic assembly of peptidoglycan (181). Mutation in either one of the alanine racemase genes does not result in o-alanine auxotrophy in S. typhimurium, because enough o-alanine is synthesized by the other enzyme. However, mutants lacking both genes require exogenous o-alanine for growth (1S1). f3-Substituted alanines inactivate both DadB and AIr racemases, but with



103

different partition ratios: 1/800 (i.e. 1 inactivation event per 800 turnovers of a,{3-elimination) for the DadB racemase and 1/160 for the Air racemase irrespective of stereoisomerism at C2 and the nature of the {Heaving group (177, 183). The nascent a-aminoacrylate produced from {3-substituted ala nines has been proposed to act as nucleophiles to capture the PLP aidimine covalently. ( l-Aminoethyl)phosphonic acid (AlaP) is an interesting inhibitor of alanine racemase; it inactivates the enzyme of Gram-positive bacteria in a time-dependent fashion, while it inhibits the enzymes of Gram-negative bacteria reversibly. The enzymes of Gram-positive bacteria (S. faecalis and B. stearothermophilus) undergo a time-dependent inactivation, which leads to stoichiometric labeling with U-[14C]-phosphonoalanine (178,184). However, intact phosphonoalanine is released from the inactivated enzyme by de naturation; the irreversible inactivation is not due to a covalent modification of the enzyme, but rather to extraordinarily slow off rates from an enzyme inhibitor binary complex. For the B. stearothermophilus enzyme, the koff is 4 x 1O-7sec-l. The molecular bases of this tight binding and selectivity for Gram-positive bacterial racemases are still unclear. Alanine racemase of S. faecalis has been purified to apparent homogeneity after a 25,000-fold increment in the specific activity from the cell extract (178). lnagaki et a1 have cloned the racemase gene of another Gram-positive bacterium, B. stearothermophilus, into E. coli, and established an efficient purification procedure for the enzyme based on its thermostability (179). Heat treatment of crude extracts from the recombinant E. coli cells raised the specific activity of alanine racemase 5-7-fold without loss in activity. Cloning of the gene for thermostable alanine racemase in E. coli was very useful for purification of the expressed gene product. The B. stearothermophilus enzyme consists of two subunits that are iden tical in molecular weight (179). Its subunit structure and some other physi cochemical properties (e.g. amino acid composition, absorption and circular dichroism spectra) are similar to those of the broad-specificity amino acid racemase from Pseudomonas putida. In contrast, however, DadB and Air enzymes of S. typhimurium and the S. faecalis enzyme are believed to be nonspherical monomers wth molecular weights of around 40,000 in their native states (63, 177). Recently, diaminopimelate epimerase (185) and glutamate racemase (186) were purified to homogeneity and shown not to contain PLP. Some earlier studies also showed that alanine racemases do not require PLP as a cofactor (175). However, recent reports clearly have indicated its presence in homogeneous alanine racemase preparations (63, 177-179). The DadB and AIr enzymes of S. typhimurium contain one mol of PLP per mol of monomer ic enzyme . The lysine residue to which PLP is inherently bound in imine linkage is located essentially at the same position from the N-terminal amino

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acid: DadB, residue 35 of 356 residues; Air, residue 34 of 359 residues. The B. stearothermophilus enzyme also contains two mol of PLP per dimeric molecule. The circular dichroic spectrum of the B. stearothermophilus en zyme shows a negative peak at 420 nm, corresponding to the absorption peak. A negative circular dichroic peak caused by bound cofactor also has been observed for the broad-specificity amino acid racemase of P. putida. This correspondence suggests that optical properties of the active sites of these racemases are similar. The B. stearothermophilus enzyme is quite stable upon heat treatment at 70°C for 80 min in 10 mM potassium phosphate buffer (pH 7.2). This high thermal stability of the enzyme thus makes it a good candidate for high-resolution X-ray analysis. Analysis of the crystal structure is in progress (187). The complete amino acid sequences of alanine racemase from four different sources [B. stearothermophilus (61); B. subtilis (60); S. typhimurium, DadB; S. typhimurium, AIr] permits comparison of the four complete sequences in order to find homologous regions in their primary structures. Despite the fact that Bacillus and Salmonella belong to Gram-positive and Gram-negative bacteria, respectively, and that the dadB (minute 36) and air (minute 91) genes map at two distinctly different regions of the S. typhimurium chromo some, the four racemase sequences display considerable homology: 74 res idues match in the four sequences and 53 residues match in three sequences compared (about 33% homology on average for the four sequences). The sequence homologies between the two racemases are calculated as 54% (B. stearothermophilus:B. subtilis); 43% (DadB:Alr); 35% (B. stearothermophi lus :DadB) ; 34% (B. subtilis:DadB); 31% (B. stearothermophilus:Alr); and 30% (B. subtilis:Alr). The extensive homology between DadB and Air strongly suggests their evolution from a common ancestor by gene duplica tion. An octapeptide containing the active-site Iysyl residue that binds PLP occurs in all four enzymes. Broad-specificity amino acid racemase of P. putida contains the same sequence. These racemases also probably evolved from a common progenitor. Faraci & Walsh have determined thermodynamic profiles for alanine race mases based on kinetic isotope effects and substrate a-hydrogen exchange with solvent (188). The mechanism of racemization by the enzyme from Gram-positive bacteria (B. stearothermophilus) appears to differ from that from Gram-negative bacteria (S. typhimurium; both DadB and Air). Galakatos & Walsh have shown that native DadB and Air racemases are digested at homologous positions by a-chymotrypsin, trypsin, and subtilisin to produce two nonoverlapping polypeptides of Mr 28,000 and 11,000 (189). Both enzymes are composed of two domains, which are linked by a hinge. The two domains remain associated after the cleavage at the interdomain bridge under nondenaturing conditions. Both clipped enzymes retain about

PYRIDOXAL

5' -PHOSPHATE

ENZYMES

1 05

3% of the original activity; the active-site geometry and secondary structure are not distorted by the proteolysis. The hinge region is conserved also in the primary structure of alanine racemases from B. subtilis and B. stearother mophilus.


a-Amino'- E-Caprolactam (ACL) Racemase ACL is a chiral heterocyclic compound synthesized in chemical industry and is used as the substrate for the industrial production of L-Iysine; L-ACL is hydrolyzed to form L-Iysine by L-ACL hydrolase (EC 3.5.2. -) of a yeast, and the unreacted D-isomer is racemized with bacterial ACL racemase . ACL racemase (EC 5.l . l .- ) is unique among racemases in acting ex elusively on ACL and a-amino-5-valerolactam. Recently, Ahmed et al stud ied the mechanism of ACL racemase ( 190). The enzyme catalyzes exchange of the a-hyrogen of the substrate with deuterium or tritium during racemiza tion in deuterium oxide or tritiated water ( 1 9 1). By tritium-incorporation experiments , the enzyme has been shown to catalyze both inversion and retention of configuration of the substrate with a similar probability in each turnover. When [a-2H]-D-ACL and unlabeled D-ACL were converted into the corresponding L-isomer by ACL racemase in water and in deuterium oxide , respectively, in the presence of excess L-ACL hydrolase , a-hydrogen (or a-deuterium) was retained significantly in the product (9 1). Therefore , a single-base mechanism has been proposed for the racemization catalyzed by ACL racemase . The ACL racemase gene has been cloned from the chromo somal DNA of Achromobacter obae, and its DNA sequence has been de termined (64). CONCLUDING REMARKS

Miles and her coworkers have extensively studied tryptophan synthase at the molecular level ( 1 92). The enzyme is now the second best understood of the PLP enzymes, but space does not permit us to review this work. The crystal structure of w-amino acid : pyruvate aminotransferase of Pseudomonas sp. F-126 also is being studied at high resolution ( 193). The structures and mechanisms of action of various PLP-dependent enzymes will continue to be studied actively. Such comparisons may offer insights into the evolution of the catalytic mechanisms used by the PLP enzymes , whose diverse reaction and substrate specificities remain intriguing. ACKNOWLEDGMENT

We thank Dr. Esmond E. Snell, Department of Microbiology, The University of Texas at Austin, for his critical reading of the entire manuscript and his kind comments and suggestions on it.

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PYRIDOXAL 5' -PHOSPHATE ENZYMES 45. Kielland-Brand, M. c . , Holmberg, S . , Petersen , J. G . L. 1 9 84 . Carlsberg Res. Commun. 49:567-75 46. Zalkin, H . , Yanofsky, C. 1 982 . J. Bioi. Chem. 257 : 1 49 1 -500 47. Henner, D. J . , Band, L . , Shimotsu, H . 1 985 . Gene 34: 1 69-7 7 48. Matsui, K . , Sano, K . , Otsubo, E. 1 986. Nucleic Acids Res. 1 4: 1 0 1 1 3- 1 4 49. Yanofsky, C . , Platt, T. , Crawford, I . P . , Nichols, B . P . , Christie, G . E . , et al. 1 98 1 . Nucleic Acids Res. 9:6647-68 50. Nichols, B. P . , Yanofsky, C. 1 979. Proc. Natl. Acad. Sci. USA 76:5244-48 5 1 . Guest, J. R . , Drapeau , G. R . , Carlton, B . C . , Yanufsky, C. 1967. J. Bioi. Chem. 242:5442-46

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