Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiae

Archives of Biochemistry and Biophysics 516 (2011) 67–74

Contents lists available at SciVerse ScienceDirect

Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi

Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiea William E. Karsten a, Zoraya L. Reyes b, Kostyantyn D. Bobyk a, Paul F. Cook a, Lilian Chooback b,⇑ a b

Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA Department of Chemistry, University of Central Oklahoma, 100 North University Drive, Edmond, OK 73034, USA

a r t i c l e

i n f o

Article history: Received 4 August 2011 and in revised form 20 September 2011 Available online 29 September 2011 Keywords: Enzymes Pyridoxal phosphate Aminotransferase Spectral studies Enzyme mechanism pH studies

a b s t r a c t The amino acid L-lysine is synthesized in Saccharomyces cerevisiae via the a-aminoadipate pathway. An as yet unidentified PLP-containing aminotransferase is thought to catalyze the formation of a-aminoadipate from a-ketoadipate in the L-lysine biosynthetic pathway that could be the yeast Aro8 gene product. A screen of several different amino acids and keto-acids showed that the enzyme uses L-tyrosine, L-phenylalanine, a-ketoadipate, and L-a-aminoadipate as substrates. The UV–visible spectrum of the aminotransferase exhibits maxima at 280 and 343 nm at pH 7.5. As the pH is decreased the peak at 343 nm (the unprotonated internal aldimine) disappears and two new peaks at 328 and 400 nm are observed representing the enolimine and ketoenamine tautomers of the protonated aldimine, respectively. Addition, at pH 7.1, of a-ketoadipate to free enzyme leads to disappearance of the absorbance at 343 nm and appearance of peaks at 328 and 424 nm. The V/Et and V/Ka-ketoadipateEt pH profiles are pH independent from pH 6.5 to 9.6, while the V/KL-tyrosine pH-rate profile decreases below a single pKa of 7.0 ± 0.1. Data suggest the active enzyme form is with the internal aldimine unprotonated. We conclude the enzyme should be categorized as a a-aminoadipate aminotransferase. Ó 2011 Elsevier Inc. All rights reserved.

Introduction The a-aminoadipate pathway for the biosynthesis of lysine in Saccharomyces cerivisiea comprises seven enzyme-catalyzed reactions, Scheme 1. The pathway begins with Claisen condensation of a-ketoglutarate (a-Kg1) and acetyl-CoA to give homocitrate (homocitrate synthase, step 1 in Scheme 1), which is followed by its conversion to homoisocitrate (homoaconitase, step 2) and oxidative decarboxylation to give a-ketoadipate (a-KA)1 (homoisocitrate dehydrogenase, step 3). Once a-KA is converted to a-aminoadipate via an aminotransferase reaction (step 4), the d-carboxylate of aminoadipate is reduced to the aldehyde (aminoadipate reductase, step 5), followed by condensation with L-glutamate to give L-saccharopine, (saccharopine reductase, step 6), which is finally converted to L-lysine and a-ketoglutarate (saccharopine dehydrogenase, step 7). With the exception of the aminotransferase, LYS genes code for the enzymes of the a-aminoadipate pathway. The aminotransferase is presumably an existing enzyme that is ‘‘borrowed’’ for the aminoadipate pathway. Because the pathway is not present ⇑ Corresponding author. Fax: +1 405 974 3862. E-mail address: [email protected] (L. Chooback). Abbreviations used: AATase, aspartate aminotransferase; Caps, 3-(cyclohexylamino)-propane-sulfonic acid; Ches, 2-(N-cyclohexylamino)ethane-sulfonic acid; Hepes, N-(2-hydroxyethyl)piperazine-N0 -2-ethane-sulfonic acid; a-KA, a-ketoadipate; a-kg, a-ketoglutarate; Mes, 2-(N-morpholine)ethane-sulfonic acid; PLP, pyridoxal 50 -phosphate; PMP, pyridoxamine 50 -phosphate. 1

0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2011.09.008

in humans, the enzymes in the pathway are potential candidates for drug development. Interruption of the lysine biosynthetic pathway is known to be a lethal event in fungi [1]. The identity of the aminotransferase that converts aketoadipate to L-a-aminoadipate is unknown. There are a number of known aminotransferases present in yeast for which the metabolic role has not been elucidated or the enzyme characterized. Our goal is to identify and characterize the aminotransferase that is likely involved in the lysine biosynthetic pathway. We are also interested in investigating the metabolic role of other aminotransferases from yeast that currently have not been characterized. A BLAST search of the yeast Aro8 protein vs. the human genome revealed the greatest similarity with a a-aminoadipate aminotransferase. This aminotransferase is also known as L-kynurenine/ a-aminoadipate aminotransferase. Based upon the BLAST sequence alignment the yeast enzyme is 27% identical and 47% similar to the human a-aminoadipate aminotransferase. The enzyme encoded by the Aro8 gene also has some similarity to human aromatic aminotransferases. The sequence similarity with the human a-aminoadipate aminotransferase suggested the aminotransferase encoded by the Aro8 gene may be a likely candidate for the enzyme that catalyzes the same reaction in the lysine biosynthetic pathway in yeast. The Aro8 gene encodes a protein with a subunit molecular mass of 56,168, which apparently exists as a homodimer [2]. On the basis of primary sequence, the Aro8 gene product is in subgroup I

68

W.E. Karsten et al. / Archives of Biochemistry and Biophysics 516 (2011) 67–74 -

O

-

O

-

O

O O O

O +

O

OH

CoASH

O

O OH

CO 2-

S (1) CoA

H -

(2)

O 2C

Mg

2+

CO 2-

(2)

(3)

O -

NADH, H +

NAD +

H

CO 2

O

O

O

O-

O

-

O

O-

α-K G H o m o c i tra t e Acetyl-CoA Homo-cis-aconitate Homoisocitrate ____________________________________________________________________________________________________________________________________________ -

-

O

-

O

-

O

O H

O

H 3N α-KG

O O

+

NADP , AMP MgPP, H +

NAD(P)H MgATP

O L-Glu NADPH

H

+

H 2O NADP+

-

O

O

O

+

H3N +

L-Glu

O

H

H 3N H 2+ N

(6)

(5)

H

(4)

-

O

O

H

O

O O-

H OL-α-aminoadipate L-saccharopine semialdehyde ___________________________________________________________________________________________________________________________________________ α-KA

L-α-aminoadipate

-

O O

α-KG NADPH

H 2O NAD +

H H 3N +

(7) L-Lysine NH 3+

Scheme 1. a-Aminoadipate pathway for lysine synthesis in fungi. The enzymes are (1) homocitrate synthase, (2) homoaconitase, (3) homoisocitrate dehydrogenase, (4) aAminoadipate aminotransferase, (5) a-aminoadipate reductase, (6) L-Saccharopine reductase, and (7) L-Saccharopine dehydrogenase. The reaction of interest is indicated in bold.

H

H

R-H 2 C-C-COO -

Lys-Enz

H 2 N +-H

H

R-H 2 C-C-COO H 2N:

N:

R-H 2 C-C-COO -

Lys-Enz

N

+

H 2N

N

H O-

R

R

Lys-Enz

H 3N

H R-H 2 C

+

COO -

N

R-H 2C

C

COO -

O-

N

H quinonoid (V) 480-540 nm

H 3N

C

+ N

H R

Lys-Enz

H 2N

+ H external aldimine (IV) ~410

Lys-Enz

+

N H O-

N

H gem-diamine (III) 325 - 340 nm

(II)

R-H 2 C-C-COO -

R

+

N

H

Lys-Enz

N

O-

R

+

N

H internal aldimine (I) ~343 nm

H 2N:

H

O-

+

Lys-Enz

+

+

NH 2 H

H 2C

O - H2O

R

+

+ N

H ketimine (VI) ~330 nm

O-

R

+ keto-acid

N

H PMP (VII) ~330 nm

Scheme 2. Proposed acid–base mechanism for the aromatic/aminoadipate aminotransferase.

of the PLP-dependent aminotransferases as classified by Mehta and Christen [3]. Aminotransferase subgroup I comprises aspartate,

alanine, histidinol-phosphate, phenylalanine, and all described aromatic aminotransferases such as the tyrosine:2-oxoglutarate

W.E. Karsten et al. / Archives of Biochemistry and Biophysics 516 (2011) 67–74

aminotransferase and phenylalanine:pyruvate aminotransferase of Pseudomonas denitrificans. In this manuscript, we report the cloning of the Aro8 gene from S. cerevisiae, and the expression and purification of the resulting enzyme. The aminotransferase derived from the Aro8 gene was characterized using initial velocity studies, pH rate profiles, and spectral studies to determine the kinetic mechanism and substrate specificity. Based upon these results we also propose a chemical mechanism for the enzyme. Materials and methods Chemical and reagents Pyridoxal 50 -phosphate, a-kg, a-KA, and L-tyrosine were purchased from Sigma. The buffers Hepes, Mes, Ches, and Caps were purchased from Research Organics. Imidazole was purchased from USB. Protein concentrations were determined by the Bradford method [4] using the Bio-Rad protein assay kit and bovine serum albumin as a standard. The affinity matrix Ni-NTA was from Qiagen. T4DNA ligase, restriction endonucleases, pfx polymerase, and the dNTP mix were from Invitrogen. The expression vector pET16b was from Novagen. Cloning of yeast Aro8 gene The cloned Aro8 gene from S. cerevisiea was obtained from the Harvard Institute of Proteomics Plasmid Repository and was contained in the plasmid pDONOR. The gene was sub-cloned into the expression vector pET16b as follows. The gene was amplified by PCR using 10 ng of plasmid DNA, 1 lM each of the forward primer 50 -GCATTACCAT-ATGACTTTACCTGAATC-3 0 and reverse primer 50 GTAATCCTCGAGGTACAAGAAA-GCTGGGTC-30 , 0.4 mM dNTPs and 2.5 units of pfx DNA polymerase. The reaction was carried out for 30 cycles as follows: 94 °C for 30 s; 53 °C for 30 s and 68 °C for 2.5 min. Prior to the start of the first cycle the DNA was denatured for 1 min at 95 °C, and at the end of the PCR, extension was continued for an extra 5 min. The PCR product was digested with NdeI and XhoI restriction endonucleases and used to sub-clone into the expression vector pET16b, previously digested at the NdeI and XhoI sites. Protein expression and purification Escherichia coli BL21-Codon-Plus (DE3)-RIL cells harboring the ARO8 gene were grown in 3 L of LB medium containing 100 lg/ mL ampicillin and 120 lg/mL chloramphenicol. When the culture reached an OD600 of 0.7–0.9, IPTG was added to a final concentration of 0.5 mM. The growth was continued at 30 °C overnight. The cells were harvested by centrifugation at 3500g and suspended in lysis buffer (50 mM potassium phosphate and 300 mM sodium chloride pH 8:0) and disrupted by sonication using a Misonix ultrasonic liquid processor at amplitude 8 for 1.5 min. The sonicated cells were centrifuged at 10,000g for 30 min. The supernatant was loaded onto a Ni-NTA column, and the column was washed with lysis buffer containing 50 mM imidazole. The protein was eluted using increasing concentrations of imidazole (100–250 mM). The enzyme was eluted at an imidazole concentration between 150–200 mM. The protein solution (15 mL) was immediately dialyzed against 500 mL of 50 mM phosphate buffer pH 7.5 for 3 h to remove imidazole. The protein solution was transferred to fresh buffer and dialysis continued for additional 2 h. At this stage it was found the enzyme displayed little absorbance in the range of 300–450 nm suggesting the enzyme as isolated contained little or no PLP. Consequently, PLP was added to the protein solution to a slight molar excess over enzyme subunit

69

concentration. Free PLP was subsequently removed by dialysis vs. 50 mM phosphate buffer pH 7.5. The resulting enzyme was concentrated by ultra-filtration to a final concentration of 10 mg/mL. The enzyme was stored at 4 °C and is stable for several weeks under these conditions; freezing and thawing causes the enzyme to precipitate. Enzyme was 95% pure based upon SDS/PAGE analysis. Three liters of bacterial culture yields 35–40 mg of pure protein. Initial velocity studies Enzyme assays were carried out using either an HP-8453 diode array or a Beckman DU640 spectrophotometer. Assays were done in 1 mL total volume using 1 cm pathlength cuvettes. The aromatic aminotransferase reaction was followed directly at 310 nm sed on a DA310 resulting from the appearance or disappearance of the keto-acid [5]. The extinction coefficients used at 310 nm were 24.5 M1 cm1 for aketoglutarate and 760 M1 cm1 for 4-hydroxyphenylpyruvate and phenylpyruvate. Initial velocity patterns were determined by varying one substrate at different fixed concentrations of the second substrate. A typical assay contained 100 mM Hepes, pH 7, fixed substrate at 10 times Km, and varied substrate from 0.5 to 5 times Km. pH studies The pH dependence of kinetic parameters was studied under conditions where one substrate was maintained at a saturating concentration and the second substrate was varied. The buffers used for the pH study were Mes (pH 6), Hepes (pH 7 and 8), Ches (pH 9) and Caps (pH 10) to cover the pH range from 6 to 9.5 at a final buffer concentration of 100 mM. The individual buffers were tested for enzyme inhibition and none was found. Mixed buffers were used to cover intermediate pH values between the pH values of the individual buffers. The pH of the reaction mixtures was measured after completion of the reaction. At each pH a substrate saturation curve was obtained varying a-ketoadipate (0.25–2.50 mM) at a fixed tyrosine concentration of 5 mM (10Km) or varying tyrosine (0.6–5.0 mM) at a fixed a-ketoadipate concentration of 8 mM and 5 lg of enzyme to obtain values for V/KEt and V/Et. Uv–visible spectral studies An HP-8453 diode array spectrophotometer was used to obtain spectra of enzyme in 1 cm pathlength cuvettes and 1 mL total volume. The concentration of the enzyme in the cuvette was 1 mg/mL (17.8 lM subunit concentration). A blank with buffer alone was recorded prior to addition of enzyme. In cases where substrates were added to the enzyme solution the volume of addition was 2 lL to avoid problems with dilution. The pH dependence of the enzyme spectrum was measured using 1 mg/mL of enzyme and 50 mM Mes, Hepes or Ches buffer titrated to the desired pH. The pH of the enzyme mixture was measured after recording the spectrum. Data processing Data were fitted using the appropriate equations developed by Cleland [6] or using the Marquardt–Levenberg algorithm [7] supplied with the EnzFitter program from BIOSOFT, Cambridge, U.K. Substrates saturation curves were fitted using Eq. (1). Initial velocity patterns were fitted using Eq. (2) for a ping pong kinetic mechanism. Data for pH profiles in which the log of the kinetic parameter decreases at low pH with a slope of 1 were fitted to Eq. (3). The data for the pH spectral titration were fitted using Eq. (4).

v¼

VA Ka þ A

ð1Þ

70

W.E. Karsten et al. / Archives of Biochemistry and Biophysics 516 (2011) 67–74

VAB K a B þ K b A þ AB

ð2Þ

log y ¼ log½C=ð1 þ H=K 1 Þ

ð3Þ

log y ¼ log½ðY L þ Y H  ðK 1 =HÞÞ=ð1 þ K 1 =HÞ

ð4Þ

0.5 0.45

In Eqs. (1), (2), v is the initial velocity, V is maximum velocity, A and B are the substrate concentrations, Ka and Kb are the Km values for substrates A and B. In Eq. (3), y is the parameter of interest, C is the pH-independent value of y, YL and YH are the pH independent values at low and high pH, respectively, H is the hydrogen ion concentration, and K1 is the acid dissociation constant for an enzyme or substrate functional group important for binding and/or catalysis.

0.4 1/v (μ μMolar/min-1)

v¼

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0

Results

1

2

3

4

5

1/L-Tyrosine (mM -1)

Initial velocity studies A double reciprocal plot of L-tyrosine vs. a-ketoadipate is shown in Fig. 1. The data in Fig. 1 were fitted using Eq. (2) for a ping pong kinetic mechanism. Kinetic parameters for several substrates of the aminotransferase are listed in Table 1. The aromatic amino acids, L-Tyr and L-Phe, have similar Km values of about 0.5 and 1.0 mM, respectively. a-Ketoadipate has the lowest Km value 0.12 mM and the highest V/KEt value of about 1  105 M1s1. Of the amino acid substrates, a-aminoadipate and L-glutamate are the poorest on the basis of V/KEt values of about 2  103 and 5  102 M1 s1, respectively. Although the V/KEt values vary by more than two orders of magnitude for the substrates listed in Table 1, the V/Et values vary by only about two-fold. At a concentration of 10 mM, neither pyruvate nor L-alanine displayed enzyme activity with the Aro8 encoded enzyme. Uv–visible spectral studies

Fig. 1. A double reciprocal plot of an initial velocity pattern done at pH 7.2 varying L-tyrosine at different fixed concentrations of a-ketoadipate. The data points are experimental and the lines are derived from a fit of the data to Eq. (2). The aketoadipate concentrations are 0.05 mM (o), 0.08 mM (D), 0.15 mM (h), and 0.3 mM (x).

Table 1 Kinetic Parameters for the Yeast Aro8 Encoded Aminotransferase. Substrate

Km (mM)

L-Phenylalanine

a

a-Ketoglutarate L-Tyrosine

a

a-Ketoadipate a-Aminoadipateb b L-Glutamate

Spectra of the aromatic aminotransferase are shown in Fig. 2 in the absence and presence of increasing concentrations of a-aminoadipate at pH 7.5. The spectrum in the absence of a-aminoadipate displays a major peak centered at about 344 nm and a broad shoul-

V/KEt (M1 s1)

V/Et (s1) 3

1.0 ± 0.1

(8.3 ± 0.8)  10

3.6 ± 0.5 0.5 ± 0.1

(2.3 ± 0.2)  103 (2.5 ± 0.4)  104

8.3 ± 0.6 12.3 ± 1.6

0.12 ± 0.03 8.8 ± 1.9 26 ± 3

(10.3 ± 1.1)  104 (2.1 ± 0.1)  103 (5.0 ± 0.3)  102

18.4 ± 1.7 12.8 ± 0.8

a Kinetic parameters were determined from an initial velocity pattern. Data were fitted using Eq. (2) for a ping pong kinetic mechanism. b The kinetic parameters were determined at a fixed saturating concentration of 4-hydroxyphenylpyruvate and the data fitted using Eq. (1).

0.18 0.16 0.14

Absorbance

0.12 0.1 0.08 0.06 0.04 0.02 0 300

350

400

450

500

550

600

Wavelength (nm) Fig. 2. Absorbance spectra from 300 to 600 nm for the aromatic aminotransferase at a concentration of 18 lM in the absence and presence of L-a-aminoadipate and 25 mM potassium phosphate, pH 7.5. The L-a-aminoadipate concentrations are (_____) 0 lM L-a-aminoadipate, (. . ..) 50 lM L-a-aminoadipate, (————) 100 lM L-a-aminoadipate, (_ _ _ _) 200 lM L-a-aminoadipate, (–– ––) 400 lM L-a-aminoadipate, and (––– –––) 900 lM L-a-aminoadipate.

71

W.E. Karsten et al. / Archives of Biochemistry and Biophysics 516 (2011) 67–74

Fig. 3. Absorbance spectra for the aromatic aminotransferase at a concentration of 18 lM at different pH values in 25 mM Mes (pH 5.93) or 25 mM Hepes (pH 6.68, 7.09 and 7.68). The pH values are (_____) pH 7.68, (. . ..) pH 7.09, (————) pH 6.68, and (_ _ _ _) pH 5.93. The inset shows the absorbance change at 400 nm as a function of pH and fitted using Eq. (4). The points are experimental and the line is derived from the fit using Eq. (4).

der around 440 nm. The 344 nm peak likely represents the unprotonated internal aldimine of PLP. On addition of a-aminoadipate the 344 nm absorbance peak progressively decreases with concomitant appearance of a new peak centered at 327 nm. The 327 nm peak likely represents the PMP form of the enzyme. The Keq for the first half reaction is equal to [F][ketoadipate]/[E][a-aminoadipate], where F is the concentration of the PMP form of the

aminotransferase. At the concentration of a-aminoadipate that gives one half the decrease in the absorbance at 350 nm, Keq is equal to [ketoadipate]/[a-aminoadipate] and [a-aminoadipate] is equal to one half [E]o, where [E]o is the initial concentration of the enzyme. From the a-aminoadipate titration data, an estimate of Keq for the first half-reaction was determined at pH 7 to be equal to 0.05.

0.14 0.12

Absorbance

0.1 0.08 0.06 0.04 0.02 0 300

350

400

450

500

550

600

Wavelength (nm) Fig. 4. Absorbance spectra from 300 to 600 nm for the aromatic aminotransferase at a concentration of 18 lM in the absence and presence of increasing concentrations of 2ketoadipate and 25 mM potassium phosphate, pH 7.5. The 2-ketoadipate concentrations are (____) 0 mM, (. . ..) 0.1 mM, (————) 0.2 mM, (– –) 0.4 mM, (– –) 0.8 mM, and (–– ––) 1.3 mM.

72

W.E. Karsten et al. / Archives of Biochemistry and Biophysics 516 (2011) 67–74

The pH-dependence of the absorbance spectrum of the aminotransferase is shown in Fig. 3. At pH 7.68 the enzyme exhibits a single peak centered at 344 nm. As the pH is decreased the 344 nm absorbance band disappears and two new peaks arise centered at 325 and 400 nm. As indicated above, the 344 nm peak likely represents the unprotonated internal aldimine of PLP. At lower pH the internal aldimine becomes protonated and the two new peaks likely represent the enolimine (325 nm) and the ketoenamine (400 nm) tautomers of the protonated aldimine. A fit of the change in absorbance at 400 nm as a function of pH using Eq. (4) gave a calculated pK for the internal aldimine of 6.46 + 0.01. A titration of the PLP form of the enzyme at pH 7.1 with increasing concentrations of a-ketoadipate is shown in Fig. 4. As the a-KA concentration increases the absorbance at 344 nm disappears with concomitant increases in absorbance at 326 and 424 nm. These two absorbance peaks are similar to the values observed at lower pH and likely represent the enolimine and ketoenamine tautomers of the internal aldimine. A binding constant for a-KA of 750 + 140 lM was calculated from a fit of the absorbance change at 424 nm vs. a-KA concentration using Eq. (1). At pH 7.8, a titration of the enzyme with similar concentrations of a-KA leads to little effect on the absorbance spectra of the enzyme (data not shown). pH studies The pH dependence of the kinetic parameters V/Et, V/KTyrEt, and V/Ka-KAEt were determined over the pH range 6.5–9.5, Fig. 5. The V/ Et and V/Ka-KAEt pH-rate profiles are pH independent. In contrast, V/KTyrEt decreases at low pH giving a slope of +1. An estimated pKa of 7.0 ± 0.1 is obtained from a fit of the data using Eq. (3). Discussion Substrate specificity As expected for an aminotransferase the double reciprocal plot of initial velocity vs. amino acid concentration at different fixed levels of a-keto-acid are parallel and best fitted to the equation for a ping pong kinetic mechanism. A screen of several potential substrates for the Aro8 aminotransferase was undertaken. Amino acids with short hydrophobic side chains, such as alanine or glycine, gave no activity, nor did the keto acid pyruvate. The aromatic amino acids L-tyrosine and L-phenylalanine are good substrates for the enzyme, consistent with the assignment of the Aro8 gene product as an aromatic aminotransferase. The two amino acids are used equally well on the basis of their V/KEt values. On the other hand, although glutamate is a substrate, it is the poorest substrate compared to the other substrates listed in Table 1. The enzyme prefers the keto-analog of L-glutamate, a-ketoglutarate, based upon a 7-fold lower Kketoglutarate compared to Kglutamate and a 5-fold greater V/KEt for a-ketoglutarate. The ability of the Aro8 encoded aminotransferase to utilize both aromatic and dicarboxylic acid substrates appears to be a common feature of this class of aminotransferase enzymes. The aromatic aminotransferase from Paracoccus dentrificans is an example. This aminotransferase can use both acidic and aromatic amino acids as substrates and crystal structures have been solved for complexes with maleate and 3-phenylpropionate [8]. Maleate forms salt bridges and hydrogen bonds with the same active site residues in the P. dentrificans enzyme as it does with aspartate aminotransferase. However, the P. dentrificans aromatic aminotransferase has the ability to switch its recognition site from a carboxylate side chain to an aromatic side chain by rearranging a set of active site residues to accommodate the aromatic amino acid. In addition, R292, which is oriented into the active site and forms a salt bridge

Fig. 5. The pH dependence of V/Et, V/KEt L-tyrosine, andV/KEt ketoadipate for the aromatic aminotransferase. The points are the experimental data and the curves are derived from a fit to Eq.3 for V/KEt L-tyrosine or a straight line using the average values of the data for V/Et and V/KEt ketoadipate.

with a carboxylate of maleate, changes position and is oriented away from the active site in the complex with 3-phenylpropionate. The yeast enzyme encoded by the Aro8 gene does not share significant similarity with the P. dentrificans aromatic aminotransferase, but similar active site rearrangements may occur to account for the ability of the yeast enzyme to use both aromatic and carboxylate amino acids as substrates. The yeast aminotransferase studied here shares significant homology with the a-aminoadipate/L-kynurenine aminotransferase II from humans and should probably be classified as such rather than as an aromatic aminotransferase. The human a-aminoadipate/L-kynurenine aminotransferase II has been reported to use 16 amino acids as substrates with a wide range of side chains and a similarly wide range of a-oxo-acids as amino group acceptors [9]. The crystal structure of the human enzyme in complex with L-kynurenine reveals nine largely hydrophobic amino acid residues in the active site that are in contact withL-kynurenine [10]. Six of these nine residues are conserved in the yeast enzyme and the remaining three are conservatively substituted. The hydrophobic pocket that accommodates the aromatic side chain of L-kynurenine in the human enzyme could potentially accommodate the aliphatic portion of a substrate such asL-a-aminoadipate. Similar interactions could be involved in the yeast enzyme. The nature of the interactions will await a structural determination. However, the ability of the yeast enzyme to efficiently use a-ketoadipate as a substrate does suggest the aminotransferase encoded by the Aro8 gene may very likely be involved in the lysine biosynthetic pathway. After the PMP form of the a-aminoadipate aminotransferase reacts with aketoadipate in vivo it needs to be regenerated to E:PMP by reaction with an amino acid. Scheme 1 casts L-glutamate in this role, however, given the relative activities of the enzyme toward the different amino acids used in the screen, one of the aromatic amino acids may be a more likely candidate. Whether the Aro8 enzyme acts alone or other yeast aminotransferases are also capable of catalyzing the reaction will require a study of other uncharacterized yeast aminotransferases.

W.E. Karsten et al. / Archives of Biochemistry and Biophysics 516 (2011) 67–74

Spectral studies The PLP form of the enzyme has a single absorbance peak centered at 344 nm, which likely corresponds to the unprotonated internal aldimine of E–PLP. The absorbance maximum is somewhat lower than that observed for the prototypical AATase, which absorbs at about 350 nm [11], or PLP in solution where the range is 350–360 nm for the unprotonated aldimine [12,13]. It is presently unclear why some of the Aro8 aminotransferase absorbance spectra are blue-shifted compared to other aminotransferases. Protonation of the Schiff base nitrogen can be followed spectrophometrically at 400 nm as the pH is decreased. The two new peaks observed at 326 and 400 nm likely represent the enolimine and ketoenamine tautomers of the protonated internal aldimine [12,14]. The pK for the aldimine in the yeast aromatic aminotransferase is about 6.5 based on the spectral pH titration and is similar to the pK for the aldimine of other aminotransferases [15]. Unlike AATase where very little of the enolimine tautomer is observed at low pH and the enolimine/ketoenamine ratio is 0.1 to 0.15 [12], for the aromatic aminotranferase there is significant absorbance corresponding to the enolimine tautomer. The enolimine tautomer with an unprotonated Schiff base and an undissociated phenolic hydroxyl group is stabilized in solutions of low dielectric constant [12,16]. This observation suggests that the active site is relatively hydrophobic, which is reasonable given the hydrophobic nature of the aromatic substrates for the enzyme. Titration of the PLP form of the enzyme with a-aminoadipate leads to a decrease in absorbance at 344 nm and an increase at 326 nm representing the PMP enzyme form [12]. This observation is consistent with the ping pong kinetic mechanism that has been found for all aminotransferases. A rapid scanning stopped flow experiment (data not shown) pushing E–PLP vs. a-aminoadipate showed only disappearance of the 344 nm peak and appearance of the 326 nm peak representing E–PMP (data not shown). No spectral intermediates along the reaction pathway were observed suggesting the steps along the reaction pathway are too fast to observe under the conditions of the experiment. Titration of E–PLP with a-KA leads to a decrease in the absorbance at 344 nm and an increase at 326 and 426 nm similar to the spectral changes observed in the pH titration of the enzyme, but with lower intensity. Results suggest that a-KA binds preferentially to the protonated aldimine form of the enzyme. An alternative possibility is that a-KA binds to the unprotonated aldimine form of the enzyme and this leads to a shift in the pK of the internal aldimine to higher pH in the complex. Compared to the spectrum of E–PLP at low pH, the E:PLP–a-KA complex displays greater absorbance at 326 nm suggesting a shift in the enolimine/ketoenamine ratio to favor the enolimine tautomer in the complex. This observation suggests the environment in the active site in the complex is more hydrophobic than E–PLP at low pH. This could be the result of closure of the active site excluding water from the site and/or neutralization of the charge on R470. Arginine 368 in AATase forms an ionic interaction with the a-carboxylate of L-aspartate in the active site, and R368 is the homolog of R470 in the Aro8 aminotransferase. Interpretation of pH profiles The V/KL-tyrosine pH profile displays one pK at low pH for a functional group that is required to be unprotonated for activity. There are no substrate pK values in this region indicating this represents a pK for a group on enzyme. The pK determined for this group (7.0) is similar to the one determined from the spectral pH titration of the enzyme (6.5). The spectral titration suggests that the observed pK is for the internal aldimine which becomes protonated below a pK of about 6.5. Taken together with the V/KL-TYROSINE pH profile data

73

the results suggest the active form of the enzyme is the unprotonated internal aldimine form of E:PLP. The pKa of 7 in the V/KL-tyrosine pH profile is not observed in the V/Et pH profile suggesting the substrate binds to only the correctly protonated form of the enzyme. No break in the V/KL-tyrosine pH profile is observed up to pH 9.5. The pK for the a-amino group of L-tyrosine, which is about 9.2 [17], is not observed in the V/KL-tyrosine pH profile. This seems to suggest that L-tyrosine with the a-amino group unprotonated binds to enzyme with equal affinity as the protonated form of the substrate. The a-carboxyl group and the aromatic side chain apparently account for the most important binding interactions with enzyme. The data also suggest L-tyrosine with an unprotonated a-amine is kinetically competent. The reaction requires an unprotonated a-amine of the substrate and a protonated internal aldimine to facilitate attack of the substrate amino group on the Schiff’s base carbon leading to the gem-diamine intermediate in the first step of the reaction. It has been proposed for other aminotransferases [15,18,19] that at pH values where the substrate binds with the a-amine protonated, the pK for the internal aldimine increases and the pK for the substrate a-amine decreases in the complex to about equal values in order to get appreciable proton transfer from the substrate amino group to the internal aldimine. Since in the current study the unprotonated form of L-tyrosine appears to be kinetically competent it suggests the pK for the internal aldimine is perturbed in the complex above pH 9. At high pH the proton cannot be donated by the unprotonated substrate but must come either from solvent or some nearby enzymatic residue. The V/Kketoadipate pH profile is pH independent over the accessible pH range in this study. There is likely a proton shared between the active site lysine formerly bound to PLP and the pyridoxamine group of PMP. A pK attributed to this diamine system has been reported at about 7.2 in the serine-glyoxylate aminotransferase from Hyphomicrobium methylovorum and the low pK was attributed to a hydrophobic environment or the vicinity of a positively charged group [20]. In the yeast aromatic aminotransferase the pK for the shared proton must be >9.5 and is more like the expected solution pK values for a lysine or pyridoxamino group of the cofactor. Potentially a pK could be observed at low pH for protonating the diamine system as proposed for AATase (18). However this pK was reported to be about pH 5.8. The low pK for the diamine system in the aromatic aminotransferase is not observed and the pK is likely below pH 6.5 similar to AATase. In AATase a break on the basic side of both substrate pH profiles at about 9.2 was attributed to the pyridinium nitrogen of the cofactor [18]. The solution pK when the phenolic oxygen of the coenzyme is ionized is about 8.4 [12]. It is likely perturbed to a value greater than 9.5 in the yeast aminotransferase by hydrogen bonding to D248 which is the homolog of D230 in the human L-kynuerenine/a-aminoadipate aminotransferase that forms a hydrogen bonding interaction with the pyridinium nitrogen of the cofactor. Proposed acid–base mechanism of the aromatic aminotransferase (Scheme 2) L-Tyrosine with its a-amino group protonated binds to the unprotonated form of the enzyme (I). With the internal aldimine unprotonated this avoids charge repulsion that could occur with a protonated aldimine. Upon binding L-tyrosine the pKa for the internal aldimine is perturbed to high pH which allows for proton transfer to occur from the a-amine of L-tyrosine to the internal aldimine (II). It appears the enzyme will also bind the unprotonated form of L-tyrosine and in this case the proton must come from solvent or a nearby enzymatic group. In step (III) attack of the unprotonated a-amine of L-tyrosine on the Schiff base carbon leads to the gem-diamine intermediate. Collapse of the gem-diamine intermediate with expulsion of the e-amine of the active site lysine

74

W.E. Karsten et al. / Archives of Biochemistry and Biophysics 516 (2011) 67–74

will form the external aldimine intermediate (IV). Abstraction of the a-proton, likely by the active site lysine, will form the quinonoid intermediate (V). Proton transfer from the e-amine of lysine to C40 will lead to the ketimine intermediate (VI) and hydrolysis of the ketimine will form keto-tyrosine and the pyridoxamine form of the enzyme (VII). Our preliminary pre-steady state results did not provide direct evidence for the intermediates suggested in the proposed mechanism, however, a good deal of evidence collected for other aminotransferases support their existence. Additional studies will be required to further define the proposed mechanism. Acknowledgments This work was supported in part by funds from an Oklahoma State Regent Grant for Higher Education (021606), P20RR016478 grant from the National Center for Research Resources (NCRR) a component of National Institute of Health (NIH), and grant 011547 from University of Central Oklahoma office of Research and Grants to L.C. The work was also supported by the Grayce B. Kerr Endowment to the University of Oklahoma to support the research of P.F.C. References [1] A. Feller, F. Ramos, A. Pierard, E. Dubois, European Journal of Biochemistry 261 (1999) 163–170.

[2] I. Iraqui, S. Vissers, M. Cartiaux, A. Urrestarazu, Molecular and General Genetics 257 (1998) 238–248. [3] P.K. Mehta, P. Christen, European Journal of Biochemistry 211 (1993) 373– 376. [4] M. Bradford, Analytical Biochemistry 72 (1976) 248–254. [5] K. Inoue, S. Kuramitsu, K. Aki, Y. Watanabe, T. Takagi, M. Nishigai, A. Ikai, H. Kagamiyama, Journal of Biochemistry 104 (1988) 777–784. [6] W.W. Cleland, Methods in Enzymology 63 (1979) 103–138. [7] C.M. Bishop, Neural Networks for Pattern Recognition, Oxford University Press, Oxford UK, 1995. pp. 290–291. [8] A. Okamoto, Y. Nakai, H. Hayashi, K. Hirotsu, H. Kagamiyama, Journal of Molecular Biology 280 (1998) 443–461. [9] Q. Han, T. Cai, D.A. Tagle, H. Robinson, J. Li, Bioscience Reports 28 (2008) 205– 215. [10] Q. Han, H. Robinson, J. Li, Journal of Biological Chemistry 283 (2008) 3567– 3573. [11] T. Yano, Y. Hinoue, V.J. Chen, D.E. Metzler, I. Miyanara, K. Hirotsu, H. Kagamiyama, Journal of Molecular Biology 234 (1993) 1218–1229. [12] C.M. Metzler, D.E. Metzler, Analytical Biochemistry 166 (1987) 303–327. [13] D.E. Metzler, in: P. Christen, D.E. Metzler (Eds.), Transaminases, Wiley, New York, 1987, pp. 60–93. [14] Y.M. Torchinsky, in: D. Dolphin, R. Poulson, O. Avramovic (Eds.), Vitamin B6 Pyridoxal Phosphate, Wiley, New York, 1986, pp. 303–327. [15] H. Hayashi, H. Mizuguchi, H. Kagamiyama, Biochemistry 37 (1998) 15076– 15085. [16] X. Zhou, M.D. Toney, Biochemistry 38 (1999) 311–320. [17] R.M.C. Dawson, D.C. Elliot, W.H. Elliot, K.M. Elliot, Data for Biochemical Research, Oxford University Press, UK, 1986. pp. 30–31. [18] D.M. Kiick, P.F. Cook, Biochemistry 37 (1983) 375–382. [19] J.F. Kirsch, G. Eichele, G.C. Ford, M.G. Vincent, J.N. Jansonios, Journal of Molecular Biology 174 (1984) 497–525. [20] W.E. Karsten, T. Ohshiro, Y. Izumi, P.F. Cook, Archives of Biochemistry and Biophysics 388 (2001) 267–275.