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407-412

EFFECTS OF REPLACEMENTOF TRYPTOPHAN-140 BY PHENYLALANINE OR GLYCINE ON THE FUNCTION OF ESCHERICHIA m ASPARTATEAMINOTRANSFERASE* Hideyuki Hayashi, Yasushi Inoue, Seiki Kuramitsu, Yoshimasa Merino* and Hiroyuki Kagamiyama* Department

of Medical Chemistry, Osaka Medical Takatsuki, Osaka 569, Japan

College,

+Department of Biochemistry, Kumamoto University Medical School, Kumamoto 860, Japan Received

,January

29,

1990

aspartate aminotransferase has been converted to SUMMARY. Trp140 of E. a Phe or Gl;y by site-directed mutagenesis. As compared to the wild-type enzyme, either of the mutant enzymes showed lo- to loo-fold increase in Km’s for natural dicarboxylic substrates, but did not show appreciable changes in Km’s for aromatic substrates. The kcat values for dicarboxylic and aromatic substrates were greatly decreased by iTrpl40 -+ Glyl mutation, but were decreased to lesser extents by [Tip140 -+ Phel mutation. These findings suggested that N(1) of Trp140 may not be essential for catalysis, but may be partly involved in the binding of the distal carboxylate group of the dicarboxylic substrates. 01990 nca&?m1c mess, Inc.

Recent X-ray crystallographic mitochondrial

(2) aspartate

aminotransferases,

studies on pig cytosolic

aminotransferases

EC 2.6.1.11

(1) and chicken

[L-aspartate:

(ASPATS 1, and subsequently

2-oxoglutarate on L

__ coli

AspAT (3) revealed that most of the active site residues are conserved in the three-dimensional

structure

of these enzymes. Trp1401 is one of these active

site residues, and is invariant

amongall the AspATs so far studied.

Accord-

*This work was supported in part by Grants-in-Aid for the Special Promotion of Science (No. 60060005) and for Scientific Research (No. 01770165) from the Ministry of Education, Science and Culture of Japan. *To whomcorrespondence should be addressed: Department of Medical Chemistry, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569, Japan. lAmin acid residues are numberedaccording to the sequence of cytosolic from pig (1).

AspAT

The abbreviations used are: AspAT, aspartate aminotransferase; HEPES, N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid; PLP, pyridoxal 5’-phosphate; PMP, pyridoxamine 5’-phosphate; W140G(F) AspAT, aspartate aminotransferase with Trpl40 replaced by Gly (Phe). 0006-291X/90$1.50 407

Copyright 0 1990 by Academic Press, Inc. AlE rights of reproduction in any form reserved.

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ing to crystallographic

data on the PLP- and Plum-forms of the enzyme and the

complex of the PLP-form

enzyme with

the indole pyridine

ring

ring

the enzymatic unclear.

of Trp140 is

(1,2).

In this

changing

this

ic properties

in a position

This situation

reaction.

a substrate

However,

communication,

suggests the precise

analogue for

stacking

the importance function

a-methylaspartate, with

the coenzyme

of the residue

of the residue

we examined the functional

role

residue

to Phe or to Gly, comparing the spectroscopic

between

the wild-type

in

is still

of Trp140 by and kinet-

and the mutant enzymes.

MATERIALS AND METHODS As described earlier (4,5), E. coli Bacterial Strains and Phage or Plasmid TY103 stramh?&ks AspAT activity was made from JM103 (6) by incorporating kanamycin, chloramphenicol, and tetracycline resistant genes into a& and Q@, and recA genes, respectively, and was used to express the mutant AspATs. The aspC gene was Site-directed Mutagenesis of Aspartate Aminotransferase excised from pKDHE19 (7) with =RI and HindIII, and recloned into M13mp18 The single-stranded DNA obtained was used for oligonucleotidephage (8). Triicosamer oligonucleotides, GC-AAC-CCA-AGC-GGC-CCGdirected mutagenesis. AAC-CAT (for W140G), and GC-AAC-CCA-AGC-TTC-CCG-AAC-CAT (for W140F) were used for mutagenesis according to the method of Taylor et al. (9). The mutant aspC genes in the double-stranded M13mp18 phages were excised with &RI and S&I and transferred into &RI&&I site of pUC19 vector (10). The resulting plasmids were verified by sequencing the entire coding frame by the method of Sanger (11) . These plasmids overproduced the mutated AspATs in -E. coli TY103. Spectrophotometric Measurements The absorption and circular dichroic spectra of the wild-type and the mutant AspATs were measured using a Hitachi spectrophotometer (model 320) and a Jasco spectropolarimeter (model J-500), respectively, at 25’ C. A buffer solution (HEPES, or potassium borate at concentration of 50 mM), containing 0.1 M KCl, was used to adjust the pH of the enzyme solution to 8.0. Stopped-flow Kinetic Studies The fast reactions of the enzymes with the substrates werestudiedatc, pH 8.0 (50 mM HEPES, containing 0.1 M KCl). by using a stopped-flow apparatus-(Union Giken, RA-1300). The apparatus had.a dead time of 1.5 ms under our operating conditions (5 Kg.cm-2 N2 gaspressure). The enzyme concentration in the reaction mixture was about 5 BM. Determination of protein concentration The concentration of the wild-type AspAT was determined s ectrophotometrically by using a molar extinction coefficient EM = 4.7 x 1OJ M-l.cm-l (PLP-form) and EM = 4.6 x lo4 M-l.cm-1 (PMPform) at 280 run and at pH 8.0. For the mutant enzymes, the values of EM were corrected for the absence of a single Trp residue (EM = 6000 M-l.cm-l)z. RESULTS The circular 250 nm region

were

gesting

the lack

spectra

within

2Kuramitsu,

dichroic

spectra

indistinguishable of gross

the visible

S. et al.;

of W140F and W140G AspATs from that

conformational region,

manuscript

which

change upon these provide

in preparation. 408

of the wild-type

information

in the 200enzyme, sug-

mutations. on the state

The of

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z 45

zE ?. “E Y

P

COMMUNICATIONS

“E

: iz

2

1

*:0

0 ” IO

b

T; -1

-2

T;

e-2

-4

G

E

200

300

400

500

(nm)

WAVELENGTH

Figure 1. Circular dichroic spectra of the wild-type () and W140F (-----) and W140G (,....) mutant enzymes. The spectra were obtained in 50 mM potassium borate buffer (pH 8.0) containing 0.1 M KC1 at pH 8.0, 25’C. [ 8 1~. mean residue ellipticity; [0 1~. molar ellipticity.

the coenzyme bound to the enzyme, were essentially

the samebetween the wild-

type and the mutant enzymes (Figure 1). The PLP-form of the wild-type

and the mutant AspATs showedan &sorption

band at 360 nm at pH 8.0. which was attributable Upon addition

of amino acids,

330 nm, which reflected

the absorption maximumshifted

from 360 nm to

the conversion of the coenzyme PLP to PMP. On the

other hand, the addition

of keto acids to the PMP-form of the enzyme was

accompanied by the spectral transamin,ation half

to the enzyme-bound PLP.

shift

reactions

from 330 nm to 360 nm. Thus, the rates of

of the wild-type

and the mutant enzymes with

amino and keto acid substrates were determined by monitoring the change in absorbance at 360 nmunder single turnover conditions (12). The kinetic wild-type

parameters thus obtained for

the half

and the mutant AspATs are summarizedin Table 1.

by Wl4OGmutation,

Km’s for oxalacetate

reactions

Either by Wl40F or

and a -ketoglutarate

by 2 orders and 1 order of magnitude, respectively,

of the

were increased

and Km’s for aspartate and

glutamate becametoo large to measure. The kcat values for the reactions with oxalacetate

and a-ketoglutarate

decreased by about 500-fold by W140Gmuta-

tion,

while the corresponding values decreased by only 4-fold

tion,

showing that W140FAspAT retained still

tence.

The kcat values for

a fairly

by W140Fmuta-

high catalytic

the two mutant enzymes in the reactions 409

compewith

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Table 1. Kinetic parameters for the transamination half reactions of the wild-type and the mutant enzymes with substratesa Substrateb/ Enzyme Aspartate Wild W140FC W140GC Oxalacetate Wild W140F

Km (mm

kcat (s-1)

4 -

500

0.035 2.8 2.6

800 210 1.4

Tryptophan Wild W140F W140G

Indolepyruvate Wild W140F W140G

(M-1.,-1)

120,000 73 0.35

-

W140G Glutamate 38 Wild W140FC W140GC a-ketoglutarate Wild 1.3 W140F W140G

bat/Km

23,000,OOO 75,000 540

700

-

600 130 1.6

22 27 33 40 46

18,000

57 0.31

460,000 6.000

59

30 2.5

880 63 2

0.092

3.0 3.3 2.6

54 17

1.1

18,000 5,200 420

aThe reaction was performed in 50 mMHEPESbuffer, pH 8.0, containing 0.1 M KC1at 25-C. h’fhe substrate concentration ranges were as follows. Wild w; 0.5-5 mMfor aspartate, a-ketoglutarate, and indolepyruvate, 5-50 mMfor glutamate, 5-25 mM for tryptophan. and 0.01-0.1 mM for oxalacetate. Mutants; 5-50 mMfor aspartate, glutamate, and aketoglutarate, 5-25 mMfor tryptophan. and 0.5-5 mM for oxalacetate and indolepyruvate. CSaturation wasnot observed.

dicarboxylic

amino acids were not measurable because the rate of these reac-

tions were not saturable with the substrates in the concentration

ranges used

in the experiments (Table -1). Aromatic amino or keto acids are fairly AspAT (13). catalytic

We thus investigated

efficiency

good substrates

still

E. coli

whether W140For W140Gmutation affected

toward tryptophan

or indolepyruvate

values for these aromatic substrates were not affected mutations.

for

In the reactions with indolepyruvate

(Table -1). appreciably

and tryptophan,

retained kcat values 31% and 8.3%, respectively,

the

The Km by these

W140FAspAT

of those of the wild-

type enzyme, while the W140Gmutation resulted in au extensive decrease in the kcat values (2% and 0.3%, respectively). 410

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DISCUSSION The results tant

presented

for the catalytic

cy (kcat/Km)

in this

function

communication

of AspAT.

was more pronounced

show that

The decrease

Trp140 is impor-

in catalytic

by W140G mutation

efficien-

than by W140F mutation

(Table 1). The effects rized

of mutation

at position

ic substrates,

but did not affect

2) The extents dicarboxy.lic

increased

the Km values

those for aromatic

of the decrease

and aromatic

are summa-

dicarboxyl-

substrates.

in kcat values

substrates

for

for the reaction

were much less

with

with

both

W140F mutation

than

W14OG mutation. The X-ray

carboxylate

crystallographic

group of

The importance ly

parameters

as follows. 1) Both W140F and W140G mutation

with

140 on kinetic

dicarboxylic

of this

supported

tion

(14) and site-directed

tion

to this

study

ing strongly

values

replacing for

than for

contribution

of this

to Arg292.

of substrates

(4,5,12) bonding

studies between

the dicarboxylic

that,

in addition

may fortify

site.

In addi-

the distal

carboxylate

(1,2,15).

The present

in a large studied,

to hydrogen-bonding

that

support-

with

of dicarboxylic

the fact

decrease

Arg292, amino or

the increase

in Km

was more pronounced for oxalacetate

cr-ketoglutarate additional

modifica-

on Arg292.

substrates

the binding

Furthermore,

was also strongchemical

has also been suggested

accompanying the W140F mutation

substrate)

hydrogen-bonded

including

all

Trpl40

to the active

the distal

Trp140 by Phe or Gly resulted

the contention with

have showed that is

of evidence,

a hydrogen

affinity

the interaction

substrates

mutagenesis

atom of Trpl40

in the binding

keto acids

lines

interaction,

showed that

(1,2,3)

hydrogen bond in binding

by several

group and N(1)

studies

(C5 substrate)

interaction

may suggest

to the binding

(C4

a larger

of C4-substrates

than that of C5-substrates. The crystallographic gradually the indole Thus,

during ring

Trp140

data have suggested

catalysis situated

may act

(1.2).

The tilt

in a position as a structural

for

that

rotates

angle appears

to be regulated

stacking

the coenzyme ring.

component 411

the coenzyme ring

with

to control

the rotational

by

Vol.

167,

No.

BIOCHEMICAL

2, 1990

movement of the coenzyme during icant

catalytic

activity

suggest

the importance

this

position

for catalytic

tion

significance

BIOPHYSICAL

RESEARCH

The present

catalysis.

was retained

Gly,

A possible

AND

by replacing

of the presence

or bulky

side chains

that a signif-

Trp140 by Phe but not by

of a bulky

aromatic

residue

at

activity3. of the presence

of an aromatic

140 for the coenzyme dynamics is now being studied

aromatic

results

COMMUNICATIONS

into

position

residue

by incorporating

at posiother

140.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Arnone, A., Rogers, P.H., Hyde, C.C., Briley, P.D., Metzler, C.M. and Metzler,D.M. (1985) in Transaminases (Christen,P. and Metzler,D.E., eds.) pp.138-155, John Wiley & Sons, New York Jansonius, J.N., Eichele, G., Ford, G.C., Picot, D., Thaller, C., and Vincent, M.G. (1985) in Transaminases (Christen,P.,and Metzler,D.E., eds.) pp.llO-138, John Wiley and Sons, New York Kamitori, S., Hirotsu, K., Higuchi, T., Kondo, K., Inoue, K., Kuramitsu, Kagamiyama, H., Higuchi, Y., Yasuoka, N., Kusunoki, M., and S M&uura, Y. (1988) J. Biochem. 104, 317-318 Hayashi , H. , Kuramitsu, S., Inoue, Y., Morino, Y., and Kagamiyama, H. (1989) Biochem. Biophys. Res. Commun. 159,337-342 lnoue, Y., Kuramitsu, S., Inoue, K., Kagamiyama, H., Hiromi, K., Tanase, S and Morino, Y. (1989) J. Biol. Chem. 264, 9673-9681 Meising, J., Crea, R., and Seeburg, P.H. (1981) Nuclic Acids Res. 9, 309-321 Kamitori, S., Hirotsu, K., Higuchi, T., Kondo, K., Inoue, K., Kuramitsu, Kagamiyama, H., Higuchi, Y., Yasuoka, N., Kusunoki, M., and S Maisuura, Y. (1987) J. Biochem. 101, 813-816 Norrander, L., Kempe, T., and Messing, J. (1983) Gene 26, 101-106 Taylor, J.W., Ott, J., and Eckstein, F. (1985) Nucleic Acids Res. 13, 8764-8785 Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33, 110-115 Sanger, F., Nicklen, S., and Coulson, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467 Cronin, C.N., and Kirsch, J.F. (1988) Biochemistry 27, 4572-4579 Yagi, T., Kagamiyama, H., Motosugi, K., Nozaki, M., Soda, K. (1979) FEBS Lett. 100, 81-84 Sandmeier, E.,and Christen, P. (1982) J. Biol. Chem. 257, 6745-6750 Kirsch, J.F., Eichele, G., Ford, G.C., Vincent, M.G., Jansonius, J.N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525

SRecently, Mattingly and Martinez-Carrion reported that W140F mutation on rat mitochondrial AspAT retained 15% of the wild-type activity whereas 11406 mutation retained only 1% when assayed under standard condition (Mattingly, J.R. and Martinez-Carrion, M., the International Multidisciplinary Conference on Vitamin B6, April 10-12, 1989, Philadelphia, Abstract P-20). 412