Steady-state kinetics and spectral properties of Corynebacterium sarcosine oxidase

630

Btochtmtca etBtophyslcaActa, 742 (1983) 630-636

Elsewer BiomedicalPress BBA31498

S T E A D Y - S T A T E K I N E T I C S AND S P E C T R A L P R O P E R T I E S OF C O R Y N E B A C T E R I U M S A R C O S I N E OXIDASE SUEKO HAYASHI a, MASARU SUZUKI b and SATOSHI NAKAMURA a a Department of B~ophystcal Chemistry, Kttasato Umverstty School of Medtcme, Sagamlhara, Kanagawa 228, and b Noda Instttute for Sclenttfic Research, Noda, Chtba 278 (Japan)

(Received August 16th, 1982)

Key words Sarcosmeoxtdase, Reactton mechamsm, Spectral change, Substrate analo~ Metal ton

The overall reaction kinetics of Corynebacterium sarcosine oxidase were investigated and the reaction was shown to follow a ping-pong, bi-bi mechanism with two substrates, sarcosine and molecular oxygen. Sarcosine analogs, such as acetate, propionate and methoxyacetate, were competitive inhibitors of the reaction. Acetate caused characteristic alterations in optical and circular dichroic spectra, indicating that the microenvironment of the substrate-binding region of the enzyme increased in hydrophobicity on binding with the substrate analog. The dissociation constants of the analogs calculated from the spectral changes were in agreement with the kinetic inhibition constants. Inorganic metallic ions were also inhibitory. Of interest was the finding that the inhibition by Hg 2+ was proportional to the square of its concentration, which suggests that at least two sulfhydryl groups are related to the catalytic activity of the enzyme.

Introduction Flavm-contaming sarcosine-oxldlzing enzymes have been purified from various bacterial and m a m m a h a n sources [1-7]. Sarcoslne oxldase (sarcosine : oxygen oxldoreductase (demethylatmg), EC 1.5.3 1) from Cyhndrocarpon chdymum M-1 [7] and sarcosme dehydrogenases (sarcosme: (acceptor) oxadoreductase (demethylatmg) EC 1.5.99.1) from Pseudomonas sp. W R F [4] were reported to contain covalently bound FAD. Sarcoslne dehydrogenase from mammalian source is also reported to contain covalently bound F A D [6]. The flavin-bound peptide of the Pseudomonas sp W R F enzyme [4] has been investigated m detad. In contrast to these enzymes, sarcosme oxtdase obtained from Corynebactertum sp. U-96 was found to contam both covalently bound and noncovalently bound F A D molecules with a molar raUo of 1 • 1 [8-10] and, m addlUon, this enzyme ts known to consist of four non-~dent~cal subunxts 0167-4838/83/0000-0000/$03 00 © 1983 Elsevier BlomedacalPress

[8]. Therefore, the possible differentiated roles of the flavms in the catalysis wdl be postulated. Although this oxadase was found to catalyze an aerobic oxidauon of sarcosme to yield glycme and formaldehyde with a concomitant formation of hydrogen peroxtde [8], no detailed kinetic investigation has so far been performed. In this regard, loneuc mvesugatlons on the ov,erall as well as i n t r a m o l e c u l a r electron-transfer m e c h a m s m s should be performed Tlus paper describes a steady-state treatment of the overall catalyzed reaction of the enzyme and also deals with some spectral properties m the presence and absence of the competmve mhlbltors.

Materials and Methods Corynebactertum sarcosme oxtdase was p u n h e d as reported [8]. Chermcals were of reagent grade and obtained from Wako Chemicals, Tokyo

631

The overall act~wty of the enzyme was measured by the oxygen uptake in 0.05 M pyrophosphate buffer, p H 8.0, at 30°C by using an 'oxygen electrode' from Yellow Springs Instruments, OH. Spectrophotometrlc measurements were made with a hlgh-sensltiwty spectrophotometer, U m o n G l k e n SM 401, and C D experiments were performed with a Union Giken Dlchrograph, Model III-J These were interfaced to a rmcrocomputer, System-77, to improve the signal-to-noise ratio. The spectra were usually measured 4-16 times repeatedly, and stored and averaged in the computer. Then the averaged spectra were recorded on the chart paper. The difference spectra were also obtained similarly. Results

Steady-state kmetws of the overall reactton The initial rate of the overall reaction was followed by measuring oxygen uptake with a Clark oxygen electrode in the presence of various concentrations of sarcosme and oxygen. Although the catalyzed reaction proceeds in the presence of water, this reaction can be kanetically treated as a two-substrate reaction, since the water concentration is always much higher than that of other substrates, sarcosine and oxygen. The results are depicted in the form of Lineweaver-Burk plots with oxygen as the varying substrate and sarcoslne as the changing fixed substrate (Fig. l a), and the

b

¢z x

ordinates of the figure were replotted in terms of the reciprocals of the sarcosme concentration (Fig. lb). The results are apparently expressed by the following equation: e0

e0

Ksar

Ko2

T = Wma----~-'b[-~ar] --~---[021

(I)

where Vmax IS the maximum reacUon rate with infinite concentraUons of both sarcoslne and oxygen, e 0 is the total enzyme concentration used, and Ksar and Ko2 are the constants obtained graplucally as the slopes of the figures The estimates values of these kmeucal constants together with the Michaehs constants are hsted in Table I Since the results were well fitted to a series of parallel straight lines, the reaction may proceed according to the ping-pong, bl-bl mechanism and, therefore, may exclude the posslbdlty of forrmng a klnetically significant ternary complex, such as enzyme-sarcosine-oxygen, from the reaction sequence.

Kmencs of the substrate-analog mhzbmon Although It IS known that m a n y sarcoslne analogs inhibit the sarcosine ox~dase activity, apparently competitively with respect to sarcoslne [11], adequate and quantitative kinetic treatments have not been carried out. Table II shows the effects of the substrate analogs on the catalytic activity. In agreement w~th other sarcosine-catalyzing enzymes, acetate, propionate and methoxyacetate were potent inhibitors, while glycine and lodoacetate did not show any significant inhibitory actwity. Detaded

TABLE I K I N E T I C CONSTANTS OBTAINED BY OVERALL REACT I O N MEASUREMENTS

10 20 "~'[Ozl (XIO3M-I)

0

5 10 ~'[$arcos,ne] (XIOZM4)

Fig 1 (a) Lmeweaver-Burk plots of the mmal velocity measured with a Clark oxygen electrode at 30°C, pH 8 0 Enzyme concentratmn, 0 08 p M Sarcosme concentratmn 1 2 mM (r-I), 1 5 mM (It), 2 0 mM (A), 29 mM (4), 5 9 mM (O), and none (@) (b) Replot of the ordinate of Fig I a against the reciprocals of corresponding sarcosme concentration

V m a , , / e o was calculated from the ordinate m Fig lb The value of Ksa r was calculated from the slope m Fig lb arid Ko2 was from a figure obtmned m slrmlar experiments Vmax/e o

1000 Wan -1

Ksa r Ko2

2 1 10 -6 M nun 2 6 10 - s M nun

K m(Sar) K m (02)

21 10 - 3 M 2 5 10 -5 M

632 TABLE II EFFECT OF SARCOSINE ANALOGS ON SARCOSINE OXIDASE ACTIVITY Acuv~ty was measured m the presence of vinous concentraUons of sarcosme analogs at 30°C, pH 8 0 Enzyme concentraUon, 0 083 I~M, sarcosme concentraUon, 5 9 mM The 50% mhlbmon of the substrate analog was determined m the presence of vinous concentrations of substrate analogs and a fixed concentrauon of oxygen (240 pM) The values of K, were, calculated from the slope m Fig 3, and figures which were obtained from samxlarexperiments as m Fig 2a for prop]onate and methoxyacetate Sarcosme analog

50% mhzbmon (mM)

K, (mM)

a n ' u n c o m p e t m v e ' type with respect to oxygen. These types of patterns are quite conststent with those of the ping-pong, bl-bi m e c h a n i s m with a n inhibitor which competes strictly with the first substrate, sarcosine, b u t has n o effect o n the interaction between the second substrate (oxygen) a n d the enzyme. The i n h i b i t i o n patterns of p r o p l o n a t e a n d methoxyacetate were essentially identical with those of the acetate i n h i b i t i o n These results are expressed b y the equation:

e0 e0 --~ = Vma---~+ ~

CH3COOH CH3CH2COOH CH3OCH2COOH NH2CH2COOH ICH 2COOH

25 35 35 > 200 _<200

29 51 54 -

steady-state analyses of the l n l u b l t i o n by these c o m p o u n d s were performed I n Fig 2a a n d b are shown typical kinetic patterns of the acetate inhibition in the forms of the Lineweaver-Burk plots with respect to sarcosine a n d oxygen concentrations, respectively. As seen in these figures, the inlubltlOn was of a 'competitive' type with respect to sarcoslne a n d of

a

"

'

" D

b

.~10 E c~ x

?

5 ~(Sarcosln]

I0 ( X 102M -1 )

0

5

10

~'Io21 (xl°3M-')

Ftg 2 L m e w e a v e r - B u r k plots for sarcosme c o n c e n t r a u o n (a) a n d oxygen c o n c e n t r a t i o n (b) m the presence of various conc e n t r a t l o n s of acetate E n z y m e c o n c e n t r a u o n , 0 083 btM, oxygen

concentration (a), 240 #M, sarcosme concentraUon (b), 5 9 mM, acetate concentration, 20 mM (t3), 10 mM (zx), 5 mM (O) and none (O) Experxmental condmons were the same as described m the legend to Fag 1

sar(+CI') 1

~

+ [02----~

(2)

where [I] a n d K, are the inhibitor c o n c e n t r a t i o n a n d lnlubltlOn constant, respectively, a n d the other n o t a t i o n s bear the same m e a n i n g s as in Eq. 1 The i n h i b i t i o n constant, K,, of the acetate was estimated from the increase in the slope of Fig. 2a (replotted m Fig. 3), a n d a value of 2.9 m M was obtained T h e v a l u e s for p r o p l o n a t e a n d methoxyaeetate were also similarly o b t a i n e d as 5 1 a n d 5.4 m M , respectively (Table II)

Effects of metal tons on the catalyttc acttvlty I n order to investigate the effects of metal tons o n sarcosine oxldase activity, the following experim e n t s were carried out The enzyme was prelnc u b a t e d with a given c o n c e n t r a t i o n of metal ion for 30 n u n , a n d then the actlvtty was measured by i n t r o d u c i n g an aliquot of the i n c u b a t e d enzyme m t o a buffer solution c o n t a m i n g 5 9 m M sarcosine a n d the same c o n c e n t r a t i o n of metal ion as that of the i n c u b a t i o n mixture Z n 2+ , Cd 2+ , C u 2+ , Pb 2+ , Hg 2+ a n d Ag z+ exhibited marked i n h i b i t i o n at a c o n c e n t r a t i o n of 1 m M The i n h i b i t i o n was reversible, since the activity was completely restored by a 250-fold dilution of the i n c u b a t i o n mixture, so that kinetics analyses can be applied to the metal ion lnlubxuon Fig. 4a and b show the kmetic patterns of the Hg 2+ i n h i b i t i o n The lnl u b i t i o n was a p p a r e n t l y competitive with respect to sarcosme, however, tt was f o u n d that the values of K A , / K A were not linear to the c o n c e n t r a t i o n of H g 2+ (Fig 5a), b u t to the square of Hg 2+ conc e n t r a t l o n (Ftg. 5b, E q n 3) This was indicative of the i n v o l v e m e n t of two Hg 2+-reactIve groups m the catalyttc sttes of the

633

0

a

o

15"

A

3 I0'

~4

lb

i

2b

-5

AcONa(XIO-3M)

i

5 IO I/(Sorcosine} (xlO z M")

i

O

5 I/(Ozl

i

I0 (xlO s M-')

]:'lg 3 Replot of a ratio of the slope wRh acetate to that without acetate ( K A , / K A ) in Fzg 2a against correspondmg acetate concentration Fig 4 Lmeweaver-Burk plots for sarcosme concentrataon (a) and oxygen concentration (b) in the presence of various concentratEons of Hg 2+ Enzyme concentration, 0082 # M , oxygen concentration (a), 240 # M , sarcosme concentration (b), 5 9 m M , Hg 2+ concentration, 4 0 # M (D), 2 0/.tM (t,), 1 0 # M ( © ) and none (@) Experimental condRlons were the same as in Fig 1

enzyme. The mhlbmon constant was 1.5 • 10 - 6 M. Tlus will be discussed later (see discussion).

Opttcal absorptton and ctrcular dtchrotsm spectra of enzyme and the effect of acetate The absorption spectra of the oxtdlzed and substrate-reduced forms of the enzyme are shown in Fig. 6. In agreement with a previous report [9], the oxidized form has absorption maxama at 276, 367 and 455 nm and a shoulder at around 290 nm,

15

15

a

probably being due to tryptophanyl residues of the enzyme. The absorption spectrum of the reduced form possessed maxima at 270 and 360 nm, and a shoulder at about 285 nm m addition to that at about 290 nm m the ultraviolet region, and a broad shoulder around the 450 nm region. No significant increase m the longer wavelength region (550 nm or longer) was observed by a static anaerobic mratzon with the substrate sarcosme. The effect of acetate, a potent competmve inhibitor (see above), was investigated by the use of

. 0:

I0

I0

3( ~

=-

02J-

2[ 5

5

0

i 1(

0

. . . .

'

'

2 4 6 [Hg 2"} ( x I O 4 M )

0

i

0

i

2

i

011"

i

4

(HgZ"] z (xlO"HM e)

Fig 5 Replot of a ratio of the slope with Hg 2÷ to that without Hg 2+ of Fig 4a against corresponding Hg 2+ concentration (a) and square of Hg 2+ concentration (b)

250

3o0

sso 3~o

46o

WAVELENGTH( nml

s~o

6o0

Fig 6 Absorption spectra of the oxidized (sohd line) and reduced (dotted line) forms of sarcoslne oxidase Enzyme concentratlon, 12 1 /aM, sarcosine concentration, (reduced form), 8 mM

634

[b /

O00E

0008

0004

0004

-O0O~

0002[

.~. *

ooo,ooo,L / 1 A,..,o..-., 250

30Q

b,J"

350 300 400 WAVELENGTH (am)

" 500

GO0

FEg 7 Difference absorption spectra of the oxEdlzed (solid hne) and reduced (dotted hne) forms of sarcoslne ox~dase caused by addEtlon of acetate Inset, double reoprocal plot of increased absorption (390 nm) versus acetate concentration Enzyme concentration, 12 1 p M , acetate concentration, 2 m M , sarcosine concentratEon (reduced form), 8 m M

a high-sensmvlty spectrophotometer. As Fig. 7 shows, acetate did not affect the absorption spectrum of the reduced form, but perturbed slgmficantly the spectrum of the oxl&zed form (solid hne). The observed difference spectrum due to the acetate ad&tton possessed poslttve extrema at around 270, 390, 460 and 493 nm, and a shoulder at around 370 nm The dissociation constant of the enzyme (OXl&zed form)-acetate complex estimated from the absorption increase at 390 nm was 1 5 mM (reset of Fig. 7b), which is m

a 7~

20

a good agreement with the value of the kinetic mtubltlOn constant, K,. Fig 8 shows the CD spectra of the oxzdlzed (thick sohd hne) and reduced (thin sohd hne) forms, respectwely The enzyme contains about 25% ordered secondary structures (our former results were erroneous [9]) but no significant &fference between the oxidized and reduced forms. The CD spectra in the near ultrawolet to visible wavelength region in the presence and absence of acetate are shown m Fig. 8a The CD spectrum of the oxadlzed form (thick sohd hne) is characterized by a posmve bands throughout the visible wavelength region and three sharp bands at around 269, 285 and 290 nm, wluch might be ascribed to aromatic amino a o d reszdues, mainly tryptophan. The addition of acetate to the oxl&zed form resulted m a difference of CD spectrum (F~g. 8, tluck dotted hne), which is essentially of the same profile as the original CD spectrum, except for a negative &chrolc band at around 350 nm (Fig 9) The dlsSoclat~on constant of the enzyme-acetate complex determined by the CD increases at 385 nm was 1 4 mM, which ~s in good agreement with the value of the kinetic constant and deterrmned by the difference absorbence spectrum. The characteristic peaks in the difference CD spectrum are, as already seen, those at 385, (430), 456 and 491 nm These may correspond, respectwely, to the optical absorption peaks at 367 and 455 nm and a hidden shoulder around 480 nm,

15 5(I 10

~

a

s 15

75

lo

50

5

25

-5

-2

-1D -50 250

300

350

3DO

~6o

s6o

o

b,~

250

300

ooo

WAVELENGTH (nm)

Fig 8 CD spectra of oxadzzed form of sarcosine oxadase z n the presence (thick dotted line) and absence (thack sohd hne) of acetate and reduced form of sarcosme oradase in the presence (than dotted hne) and absence (thin solid line) of acetate (a) Enzyme concentratzon, 6 l /~M, acetate concentratEon, 5 m M , sarcoslne concentratEon (reduced form), 4 m M (b) Enzyme concentratzon, 21 /~M, acetate concentratzon, 9 9 mM, sarcosine concentration (reduced form), 8 m M

o

A xJ

350 300

480

500

600

WAVELENGTH Inrn)

Fig 9 Dzfference C D spectra of the oxadlzed form of sarcosme oxtdase Inset, double recEprocal plot of observed increased elhptEoty (385 nm) versus corresponding acetate concentratlon Experimental c o n d m o n s were the same as described m the legend to FEg 8

635 wtuch apparently is often observable with various flavoprotelns. Shoulders m the C D spectrum at around 370 and 430 n m were also recognized, which are only poorly detectable in the optical absorption spectrum. The CD spectrum of the substrate-reduced form is shown in Fig. 8 (than sohd hne), which was characterized by the &chrolc bands at 489 nm and negative band at 415 nm, and was completely devoid of the posmve bands observed in the oxidized form in the wslble region. The addition of excess acetate to the reduced form, m contrast to the ora&zed form, &d not cause any difference m C D spectrum m the near ultraviolet and ws~ble wavelength region (Fig. 8, thin dotted line), implying that there was no detectable interaction between the inhibitor and the reduced enzyme. These observations are quite consistent wah the kinetic results that acetate competes strictly with the substrate to the oxt&zed enzyme. Discussion Quantltatwe kinetic analyses have revealed that the overall reaction of Corynebactenum sarcoslne oxadase follows a ping-pong, bl-bl mechamsm with the two substrates, sarcosme and molecular oxygen. In analogy to the reaction of fungal glucose oxldase-catalysls [12-14], the reaction sequence of the

S Eox

P + HCHO F spontaneously P' 0 2

Eox S ~ Erea P'

H ~O2

Ered Ered 02 ~ Eox H202

Eox

where Eox and Erea are the oxidized and reduced forms of the enzyme, respectively, P' is the intermediary reaction product, C H 2 = N - - C H 2 C O O H , whach yields the final product glyclne, P. S, substrate. Sarcosme-analog compounds mchadmg acetate inhibited the catalyzed reaction compeutlvely with respect to sarcosine, and inorganic metalhc ions, usually considered to attack SH residues in a protein, also inhibited the reaction.

In the presence of a sarcoslne-analog mlub~tor, an equilibnuum relatmnship is added to the above equations:

Kt Eo~+I~Eox

I

In the case of Hg 2+ ad&tlon, the equdlbnum is Eox + 2Hg 2÷ ~ Eox. (2Hg 2÷ ) and the derived rate equation is eo

eo

Ksar [

V - - Vma~ - I ' - ~ - ~ / 1

[Hg2+] 2 1

'q

K?

Ko~

] " 4 -[02] --

(3)

The lnhlblt~on constants were already shown as Eqns. 1-3. The observation that Hg 2+ mlubltlon was proporuonal to the square of the concentration lmphes that (1) the actwe site may contain reactive SH groups and (2) the number of SH groups may not be smaller than two. However, thas possiblhty does not necessardy exclude an involvement of other reactwe amino acid residues from the actwe site region of the present enzyme. Tlus problem is now under investigation. The optical absorption spectrum of the enzyme was of a typical flavoproteln, and the CD spectrum of the OXl&zed form was essentially slmtlar to those of choline oradase of Alcahgenes species [15], characterized by the positive elhptlclty throughout the visible wavelength regton. It should be noticed that the OXl&zed form possessed no detectable shoulder m the 480-490 nm region, which is usually observed w a h flavoprotelns and considered as an ln&cat~on of the environmental hydrophobic~ty of the flawn moieties. Interestlngly, this band was clearly seen in the CD spectrum of the oxidized form (Fig. 8). Since the vlbronic peak is evident, the flavln moiety of this enzyme is rather deeply buried into the hydrophobic environment of the enzyme protein. Furthermore, on ad&tlon of the competluve lnhabltor, acetate, the shoulder of the opucal absorption in 490 nm region became apparent (F~g. 7), indicating that the environment of the flavm moiety increased hydrophobloty to a slgmficant degree.

636 The effect of acetate on the C D s p e c t r u m was also r e m a r k a b l e (Fig. 9) the elliptlcity in the visib l e region increased generally throughout, a n d the p e a k s b e c a m e m o r e p r o f o u n d on a d d i t i o n of acetate A further increase in these spectral p r o p erties on acetate a d d i t i o n l m p h e s an reduced-fit c o n f o r m a t i o n a l change m the active site region N o e x p e r i m e n t a l evidence was so far o b t a i n e d for b i n d i n g acetate with the r e d u c e d form of the enzyme b y the C D m the visible or n e a r - u l t r a w o l e t wavelength regton. Tlus is c o m p a t i b l e with the kinetically o b t a i n e d i n h i b m o n m e c h a n i s m as well as with the a b o v e - m e n t i o n e d c o n f o r m a t l o n a l changes. A s r e p o r t e d a l r e a d y [8-10], Corynebactenum sarcosme oxidase is a u m q u e f l a v o p r o t e l n h a w n g one covalently a n d one n o n - c o v a l e n t l y - b o u n d F A D molecule in a single p r o t e i n molecule T h e e n z y m e is also unique, since it consists of four n o n - i d e n t i cal subumts. The covalently b o u n d F A D is k n o w n to a t t a c h to one of these subunlts ( s u b u n l t B, m o l e c u l a r weight a b o u t 44 000). T h e presence of two differently b o u n d F A D molecules m a single p r o t e i n molecule m a y indicate the d~stlnct roles of these flawn molecules in the catalytic activity of the enzyme. A l t h o u g h whether subunlt B also carries the n o n c o v a l e n t l y b o u n d F A D is presently unclarlfied, the c o e n z y m e m a y be l o c a t e d sterically close to the covalently b o u n d F A D so as to faclhtate electron t r a n s p o r t b e t w e e n the two F A D molecules T h e active site region of this enzyme may, therefore, involve these electron-transferring coenzymes, the substrate-bin d l n g site, a n d the site for interacting with molecular oxygen. T h e possible residues for the s u b s t r a t e b i n d i n g are the reactive S H groups. As a l r e a d y seen in Ftg. 6, the r e d u c t m n of the e n z y m e b y the substrate d i d not show any detecta-

ble intermediates. This suggests that the l n t r a m o lecular electron transfer is extremely fast as comp a r e d with the l n t e r m o l e c u l a r electron transfer, a n d that lonetlcally no distinction could be d r a w n b e t w e e n the two flavln molecules. M o r e detailed studies b y m e a n s of r a p i d kinetics will b e required to clarify tbas p r o b l e m . It is of p a r t i c u l a r interest f r o m the viewpoint of m o l e c u l a r evolution that tlus enzyme might be r e g a r d e d as a m o d e l for two electron-transfer protelns u n i t e d together to form a single ' m o r e - d e v e l o p e d ' protein. F u r t h e r evidence is needed.

References 1 Fnsell, W R and Mackenzae, C G (1962) J Blol Chem 237, 94-98 2 Hoslons, D D and Bjur, RA (1964)J Blol Chern 239, 1856-1863 3 Patek, D R and Fnsell, W R (1972) Arch Blochem Blophys 150, 347-354 4 Pinto, J T and Fnsell, W R (1975) Arch Blochem Blophys 196, 483-491 5 0 k a , I, Yoslumoto, T, Rakatake, K, Ogucht, S and Tsuru, D (1979)Agnc Blol Chem 43, 1197-1230 6 Sato, M, Otusba, N and Yagl, K (1979) Blochem Blophys Res Commun 87, 706-711 7 Mort, N, Sano, M, Tara, Y and Yamada, H (1980) Agnc Blol Chem 4, 1391-1397 8 Suzulo, M (1981)J Blochem 89, 599-607 9 Hayashl, S, Nakamura, S and Suzuki, M (1981) Blochem Blophys Res Commun 96, 924-930 10 Hayashl, S, Suzulo, M and Nakamura, S (1982) Blochem Int 4, 617-620 11 Fnsell, W R and Mackenzie, CG (1955) J Blol Chem 217, 275-285 12 Nakamura, S and Ogura, Y (1968) J Blochem 52, 214-219 13 Gibson, Q H , Swoboda, B EP and Massey, V (1964) J Blol Chem 239, 3927-3934 14 Nakamura, S and Ogura, Y (1968) J Blochem 63, 308-316 15 Ohta-Fukuyama, M, Mlyake, Y, Shlga, K, Nlsluna, Y, Watan, H and Yamano, T (1980) J Blochem 88, 205-209