Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid

Biochimica et Biophysica Acta 952 (1988) 297-303

297

Elsevier BBA33054

Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid Kaoru Sakai, Keiji Hasumi and Akira Endo Department of Agricultural and Biological Chemistry, Tokyo Noko University, Tokyo (Japan)

(Received24 March 1987) (Revised manuscript received2 November 1987)

Key words: Glyceraldehyde-3-phosphatedehydrogenase;Enzymeinactivation; Koningicacid Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogense (D-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD +, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation (kapp) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate (k 3) was 5.7- 1 0 - 2 s - 1, with a dissociation constant for inactivation (Kinact) of 1.6 /tM. The inhibition was competitive against glyceraldehyde 3-phosphate with a K i of 1.1 /~M, where the K m for glyceraldehyde 3-phosphate was 90/~M. Koningic acid inhibition was uncompetitive with respect to NAD +. The presence of NAD + accelerated the inactivation. In its absence, the charcoal-treated NAD +-free enzyme showed a 220-fold decrease in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD +. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD +-enzyme complex.

Introduction

Glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating) EC 1.2.1.12) of rabbit muscle and yeast has been studied extensively. It is a tetramer, composed of four identical polypeptide chains that contain sulfhydryl groups es-

Correspondence: A. Endo, Department of Agricultural and BiologicalChemistry,Tokyo Noko University,3-5-8 Saiwaicho, Fuchu-shi, Tokyo, 183 Japan.

sential to their catalytic activity [1]. Koningic acid (heptelidic acid) is a sesquiterpene antibiotic with an epoxide moiety (Fig. 1). It has been shown to have antibiotic activity against anaerobic bacteria such as Bacteroides and a cytostatic effect on mouse L1210 cells [2]. In addition, koningic acid was also shown to be a specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase [3]. In the present study, a mechanism for koningic acid inhibition of rabbit muscle glyceraldehyde-3phosphate dehydrogenase was studied. The results suggested that koningic acid binds irreversibly to the sulfhydryl groups, possibly at the catalytic sites of the enzyme.

0167-4838/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (Biomedical Division)

298

o~"

o

/IL,,, " H3C

C00H

CH3

Fig. 1. Structureof koningic acid (heptelidic acid).

Materials and Methods

Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (crystalline suspension in ammonium sulfate) was purchased from Boehringer-Mannheim, which was homogeneous as judged from sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. The purity of active enzyme was 96%, as determined by titration of highly reactive cysteine at the active site of enzyme as described by Ellman [5]. The enzyme was dialyzed against 30 mM Tris-HC1 (pH 8.5) containing 1 mM dithiothreitol and 1 mM EDTA prior to use. The dialyzed preparations of enzyme had A280:A260 ratios of 1.24-1.27, corresponding to 2.1-2.3 mol of NAD + per mol of the enzyme. The removal of enzyme-bounds NAD + was carried out by charcoal treatment, essentially as described by Krimsky and Racker [4]. 10 mg of enzyme dissolved in 1 ml of 0.5 mM EDTA was applied to a column, constructed with a Pasteur pipet and 50 mg of activated charcoal, and elution was carried out with 0.5 mM EDTA at 4°C. The charcoal-treated enzyme preparations had A280:A260 ratios of 1.90-1.96, indicating that the enzyme preparations are essentially NAD +-free. Preparation of [ 3H]koningic acid Trichoderma koningii M3947 was aerobically grown at 25 °C in a 100 ml Sakaguchi flask containing 10 ml of culture medium composed of 3% malt extract, 2% glucose and 0.1% polypeptone. On the 2nd, 3rd and 4th day, 1, 2 and 2 /~Ci (2 Ci/mmol) of [3H]acetate was added to the culture, respectively. On the 5th day, the culture was acidified with HC1 (pH 3) and extracted with 10

ml of ethyl acetate. The solvent layer was evaporated to dryness under reduced pressure. From the resultant residue, [3H]koningic acid was isolated by preparative thin-layer chromatography, using silica gel plates (Merck, 60 F-254) which were developed in a solvent system of dichloromethane/acetic acid (95:5) and further purified by preparative high-performance liquid chromatography (Japan Spectroscopic Co., Finepak Sil C18 ) using a solvent system of acetonitrile/0.1% phosphoric caid (50:50). On analytical thin-layer chromatogrpahy, [3H]koningic acid showed one radioactive spot which comigrated with koningic acid (R v = 0.42). Further, [3H]koningic acid was radioactively homogeneous on analytical high-performance liquid chromatography. The purified [3H]koningic acid had a specific radioactivity of 6.2- 104 dpm/nmol.

Other materials Rabbit muscle phosphoglycerate kinase was obtained from Boehringer-Mannheim. DL-Glyceraldehyde-3-phosphate was prepared from the barium salt of DL-glyceraldehyde 3-phosphate diethylacetal (Sigma). The concentration of D-isomer was determined enzymatically according to the method of Sigma. [3H]Acetate (sodium salt) was purchased from New England Nuclear and 5,5'-dithiobis(2nitrobenzoic acid) was from Waco Pure Chemicals (Japan). Koningic acid was isolated from culture broth of T. koningii M3947 as described previously [3]. All other chemicals were of the highest grade from commercial sources. Assay of enzyme activity Glyceraldehyde-3-phosphate dehydrogenase activity was assayed spectrophotometrically according to the method of Duggleby and Dennis [6]. The reaction mixture (3 ml) contained 135 mM Tris-HC1 (pH 8.5) 3.3 mM cysteine-HC1, 1 mM potassium phosphate, 1 mM NAD +, 0.2 mM o-glyceraldehyde 3-phosphate, 0.2 mM EDTA, 0.2 mM MgC12, 0.5 mM ADP, 6.5-6.8 nM of glyceraldehyde-3-phosphate dehydrogenase, and 5 units of phosphoglycerate kinase. Reaction was started by adding glyceraldehyde 3-phosphate. NADH formation was monitored by the initial increase in absorption at 340 nm using a Hitachi 320 spectrophotometer.

299 Where indicated, glyceraldehyde-3-phosphate dehydrogenase (19.4 nM) was preincubated at 25 ° C for 0-40 min in a mixture (total volume 1 ml) containing 0.2 mM EDTA, 0.2 mM MgC12, 3.3 mM cysteine-HC1, 3 mM N A D + and 135 mM Tris-HC1 (pH 8.5) in the presence of a varying concentration of koningic acid. Dithiothreitol (1 mM) or cysteine (3.3 mM) contained in the reaction mixture had no effect on the inhibitory activity of koningic acid, and the nonenzymatic reaction between these thiols and koningic acid was not observed under these conditions. The concentration of glyceraldehyde-3-phosphate dehydrogenase was determined by taking the molecular weight of the enzyme as 144000 [7,8],

[3H]Koningic acid binding to glyceraldehyde-3phosphate dehydrogenase The reaction mixture (1 ml) for the determination of [3H]koningic acid binding to glyceraldehyde-3-phosphate dehydrogenase contained 19.4 /~M of the enzyme, 1 mM EDTA, 1 mM dithiothreitol, 30 mM Tris-HC1 (pH 8.5) and varying amounts of [3H]koningic acid (ranging from 0 - 7 moles per mole of enzyme). After incubation at 25 ° C for 5 min, aliquots (10 /~1) were removed, diluted, and assayed for enzyme activity as described above. Other aliquots (0.5 ml) were mixed with 0.5 ml of ice-cold 10% trichloroacetic acid. The resultant precipitate was collected on a membrane filter with a pore size of 0.45 /~m and washed thoroughly with cold 5% trichloroacetic acid and then with methanol. The membranetrapped materials were dissolved in 3 ml of 0.1 M N a O H and counted for radioactivity in a toluenebased scintillation fluid containing 30% Triton X-100. The amount of [3H]koningic acid bound to glyceraldehyde-3-phosphate dehydrogenase (moles of koningic acid per mole of enzyme) were determined by taking the molecular weight of the enzyme as 144000 [7,8]. For a blank assay, the enzyme, which had been treated in boiling water for 2 min, was used.

Titration of sulfhydryl groups Glyceraldehyde-3-phosphate dehydrogenase was dialyzed against 1 mM EDTA and 30 mM Tris-HC1 (pH 8.5). The enzyme (20.5 /~M) was preincubated at 2 5 ° C for 5 min in a mixture

containing 1 mM EDTA, 30 mM Tris-HC1 (pH 8.5) and varing amounts of koningic acid. Aliquots (10 /~1) were pooled for assaying enzyme activity. Other aliquots (0.2 ml) were mixed with 3 ml of 6 M guanidine hydrochloride (pH 8.5) and the mixtures were incubated at 25 ° C for 5 min at which time 20 /~1 of 1 M 5,5'-dithiobis(2-nitrobenzoic acid) containing 0.1 M sodium phosphate buffer (pH 7.0) was added to the mixture [9]. Absorption was monitored for approx. 10 min at 418 nm. The pH of the final mixture was 8.5. A control with no koningic acid and two blanks, one with koningic acid and no enzyme and one with no koningic acid and no enzyme, were run simultaneously. The absorption coefficient at 418 nm of the 5-thio-2nitrobenzoate anion obtained by titration of cysteine was found to be 2.7 • 10 4 M -1 • cm -1 at pH 8.5.

Other methods Protein was determined by the method of Lowry et al. [10] with bovine serum albumin as a standard. High-performance liquid chromatography (HPLC) of glyceraldehyde-3-phopshate dehydrogenase and its complex with koningic acid was carried out using a TSK Gel 3000 SW column (Toyo Soda Manufacturing Co., Japan). Results

Time-dependent irreversible inhibition Preincubation of koningic acid with charcoaltreated glyceraldehyde-3-phosphate dehydrogenase caused increasingly greater inhibition as the time of exposure to the enzyme increased. This is shown in Fig. 2, where the relative enzyme activity is plotted against the preincubation time with koningic acid. Enzyme activity was expressed relative to a blank to correct for spontaneous inactivation of the enzyme and competitive inhibition by koningic acid. In the experiment shown in Table I, concentrations of koningic acid at enzyme assay were reduced to a varying extent, as compared with those at preincubation. As indicated, inhibition was dependent on the concentration of koningic acid at preincubation. No significant restoration of inactivated enzyme could be obtained by decreasing koningic acid concentration at enzyme assay.

300 TABLE I

1.0 ,iiKoningic a c i d ( p M )

IRREVERSIBLE INHIBITION OF RABBIT MUSCLE GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

0.5

i"

® 0.2

~ o.~ n-

0.05 J

i

I

0

20

40

Incubation

60

8,0 100 120

Enzyme (19.4 nM) and koningic acid were preincubated at 25 ° C for 40 min in 135 mM Tris-HC1 (pH 8.5) containing 0.2 mM EDTA, 0.2 mM MgCI 2 and 3.3 mM cysteine-HC1, and, after preincubation, the concentration of koningic acid was diluted at enzyme assay as indicated, and the remaining activity was determined. Each value represents the mean of duplicate determinations. Koningic acid (#M)

Enzyme activity

Inhibi-

at preincubation

at enzyme assay

(#mol NADH/min per mg protein)

tion (%)

0 0.09 0.09 0.09 0.09

0 0.06 0.04 0.02 0.01

26.9 6.8 8.7 10.1 9.8

0 75 68 62 64

time (sec)

Fig. 2. Time-dependent irreversible inhibition of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid. Charcoal-treated enzyme (19.4 nM) was preincubated at 25 o C with various concentration of koningic acid (as indicated) in the presence of 3 mM NAD +, and the remaining activity was determined. In blank assays, enzyme was preincubated in the absence of koningic acid, which was added to the mixture at enzyme assay. The relative activity of enzyme, the ratio of the remaining activity in the presence of koningic acid at preincubation to that of a blank, was plotted vs. preincubation time in a semilogarithmic graph. Each value represents the mean of duplicate determinations.

where E is the enzyme; I is the inhibitor; E. I is the reversible enzyme-inhibitor complex; E-I is the irreversible complex of enzyme and inhibitor; Kinac t is the dissociation constant of E. I; and k 3

0.03

"3U

ID

0.02

v

Effect of koningic acid concentration As shown in Fig. 2, inactivation of charcoaltreated glyceraldehyde-3-phosphate dehydrogenase by koningic acid followed pseudo-firstorder reaction kinetics, the rate of which directly related to the inhibitor concentration. Using the slopes of the straight lines, the values of apparent rate constant for inactivation (kapp) were calculated. The dependence of k~pp on the concentration of koningic acid appears to saturate (Fig. 3) characteristics of a reversible binding process between koningic acid and enzyme prior to irreversible inactivation: Kinact

E+I

~

k3

E-I~E-I

Q. O. 0 ,.X

~

0.01

/

/.

60

•

40

l

e°e

I

I

o

1

i

I

2

(Koningic ocid)-I (}.IM-1) i

I

i

o.5 1.o 1.5 Koningic acid (pM)

i

2.0

Fig. 3. Inactivation rate as a function of koningic acid concentration. The pseudo-first-order rate constant (kapp) was plotted as a function of koningic acid concentration. Charcoal-treated enzyme (19.4 nM) was preincubated with varying concentrations of koningic acid under the conditions given in the legend to Fig. 2. The inset is a double-reciprocal plot of rate constant vs. koningic acid concentration according to the Kitz-Wilson equation [11]. Each value represents the mean of duplicate determinations.

301

is the inactivation rate constant at infinite inhibitor concentration. The double-reciprocal plot of kapp vs. the concentration of inhibitor [I] is shown in the inset of Fig. 3 according to the Kitz-Wilson equation [11]: 1

Kinact

kap p

k3

Koningic ocid (pM) •

.E

1 + 1 [11 k 3

From the slope and intercept of the plot, the kinetic constants, Kinac t = 1.6 /~M and k 3 = 5.7. 10 -2 sec -1, were calculated. Effect of substrates concentration The glyceraldehyde-3-phosphate dehydrogenase activity was assayed in the presence of various concentration of koningic acid and at different concentration of substrates. The data were plotted according to the Lineweaver-Burk equation. The inhibition of glyceraldehyde-3-phosphate dehydrogenase by koningic acid was competitive with respect to the substrate glyceraldehyde 3-phosphate (Fig. 4A). These data gave a K m value of 90 #M and a K i value of 1.1 /xM. The latter is comparable to the g i n a c t value of 1.6 #M (described above). The ratio of K i / K m of 1.2.10 -2 demonstrates that the affinity of koningic acid to the active site is 82-times higher than that of glyceraldehyde 3-phosphate. Koningic acid inhibition appeared to be uncompetitive with respect to NAD + with a K i value of 2.0 #M (Fig. 4B). These results indicate that koningic acid binds predominantly to the NAD +-enzyme complex [12]. Koningic acid showed noncompetitive inhibition against another substrate inorganic phosphate (Fig. 4C). To see the effect of NAD + on the inactivation of enzyme by koningic acid, charcoal-treated enzyme (19.4 nM) was preincubated with 0.5/~M of koningic acid in the absence or presence of 3 mM NAD ÷. After incubation for 0-30 min, the relative activity was determined as described above and the apparent rate constant for inactivation was calculated from the straight line of the semilogarithmic plot of the relative enzyme activity vs. preincubation time. The NAD÷-free, charcoaltreated enzyme showed low reactivity towards koningic acid. The apparent rate constant for in-

60

A

-_

E

~

d:

_//o

~

1

o

20 ~ , o ~ o ( flag)- 1 CraM_l)

,

20 °~o

B

T._. ao -

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~

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C

"6

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(NAD.)-I (ram -1) t

50 t

i

100 I

.A" 6f.:

-

.o 0

(pi)-I

?

4

(mM -1)

6

8,

Fig. 4. Lineweaver-Burk plots for inhibition of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid. Charcoal-treated enzyme was used at a concentration of 6.5 nM. Enzyme was assayed for 5 s at 25 ° C with the varying concentration of koningic acid (0, 1, 2 and 3 /~M) In (A) glyceraldehyde 3-phosphate (G-3-P) was varied as indicated and N A D + concentration was fixed at 0.2 mM. In (B) N A D ÷ was varied as indicated. In (C) inorganic phosphate (Pi) was varied as indicated and the N A D + concentration was fixed at 0.2 mM. Each value is the average of two assays.

activation (/£app) was 5.8.10 -5 s -1. In the presence of 3 mM of NAD +, the marked inactivation of enzyme was observed and the inactivation rate constant ( k a p p = l . 3 " 1 0 -2 s -1) was 220-fold higher than that for NAD+-free enzyme. The acceleration of inactivation of NAD + was ascribed to ordered binding of first NAD + and then koningic acid.

302

Stoichiometry of koningic acid binding The s t o i c h i o m e t r y of the b i n d i n g of koningic acid to g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o genase is detailed in Figs. 5 a n d 6. W i t h increasing a m o u n t s of [3H]koningic acid, e n z y m e activity decreased, whereas the b i n d i n g of [3H]koningic acid to the enzyme increased. A t higher c o n c e n t r a tion of koningic acid, enzyme activity was c o m pletely inhibited a n d approx. 2 mol of [3H]koningic acid p e r mol of enzyme molecule were incorp o r a t e d (Fig. 5). In the e x p e r i m e n t s shown in Fig. 6, enzyme was i n c u b a t e d with various a m o u n t s of n o n r a d i o a c t i v e koningic acid a n d the n u m b e r of r e m a i n i n g thiols was determined. T h e results showed that 2 mol of i n h i b i t o r is required to inactivate the e n z y m e tetramer. F u r t h e r m o r e , the loss of activity was associated with a loss of thiol groups a n d a m a x i m u m of 2 mol of thiol g r o u p was modified, suggesting that the enzyme was catalytically inactive when two thiol groups of the enzyme were m o d i f i e d b y koningic acid. M o r e than 2 mol of koningic acid could not b e i n c o r p o r a t e d into the p r o t e i n even when enzyme was i n c u b a t e d with excess [3H]koningic acid for long p e r i o d s of time. H i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y indic a t e d that b o t h the m o d i f i e d a n d u n m o d i f i e d en-

//---

2 -o

:>, > q.. u 0 ®

A

50

E

"©

\ O

LIJ

0 I

I

I

I

0

I 2 3 Bound 3H-koningic acid or modified thiol groups (mollmol tetramer) Fig. 6. Stoichiometry of koningic acid inhibition. The experiment was performed in two parts. (A) [3H]koningic acid binding to glyceraldehyde-3-phosphate dehydrogenase and the remaining enzyme activity were determined as described in Fig. 5. The results are plotted as activity vs. the moles of [3H]koningic acid per mole of enzyme (O). (B) Enzyme was preincubated under the same conditions as described in part (A), except that [3H]koningic acid was replaced by unlabeled koningic acid. The remaining sulfhydryl groups and enzyme activity were determined. The native enzyme was shown to have 15.9 mol of sulfhydryl group per mol of enzyme. The results are plotted as activity vs. the moles of modified thiol groups per mole of enzyme (zx). Each value represents the mean of duplicate determinations. These experiments were performed twice with similar results.

-100

® o E

o~

o..-..-,.'~,'°

8 0 00 >,

~

oo

--

-~E

40

~

0

o 0

100 v

1

2

3H-Koningic acid

3

J~

I 1/1

4

0 "'

7

zymes r e m a i n e d as tetramer. Thus, there was little d e t e c t a b l e change in the gross c o n f o r m a t i o n of the e n z y m e a c c o m p a n y i n g the r e a c t i o n with koningic acid ( d a t a n o t shown). W h e n [3H]koningic acidenzyme c o m p l e x was s u b m i t t e d to S D S - p o l y a c r y l a m i d e gel electrophoresis, r a d i o a c t i v i t y c o m igrated with the s u b u n i t p r o t e i n ( d a t a n o t shown). These o b s e r v a t i o n s suggested that [3H]koningic acid b o u n d covalently to the e n z y m e protein.

(mol/moltetramer)

Fig. 5. [3H]Koningic acid binding to glyceraldehyde-3-phosphate dehydrogenase and enzyme activity. Enzyme (19.4 /~M) was preincubated at 25 °C for 5 min with varying concentrations of [3H]koningic acid. [3H]Koningic acid binding (O) and the remaining enzyme activity (©) were determined. Each value represents the mean of duplicate determinations. This experiment was performed twice with similar results.

Discussion

R a b b i t muscle g l y c e r a l d e h y d e - 3 - p h o s p h a t e deh y d r o g e n a s e was i n h i b i t e d with c o n c e n t r a t i o n s of approx. 10 - 6 M of koningic acid (Fig. 2). These c o n c e n t r a t i o n s are c o m p a r a b l e to those for p e n t a -

303

lenolactone, a sesquiterpene antibiotic with an epoxide structure in its molecule [13,14]. The inhibitory activity of these two antibiotics are, therefore, by far higher than other irreversible inhibitors, including iodoacetate, N-ethylmaleimide, iodoacetamide, arene oxides and glycidol phosphate which are active at concentrations of 10-2_10 4 M [15-17]. Koningic acid irreversibly inactivated glyceraldehyde-3-phosphate dehydrogenase in a time-dependent, pseudo-first-order manner. However, reversible enzyme inhibitor complex (E. I) is formed prior to the formation of irreversible complex of enzyme and inhibitor (E-I). Thus, the inhibition of the enzyme by koningic acid is competitive with respect to glyceraldehyde 3-phosphate. These findings suggest that koningic acid is an active-site-directed inhibitor. The inactivation of enzyme by koningic acid was accelerated by the presence of NAD ÷. It has been shown that NAD ÷ plays a positive or negative effector of enzyme activity dependent on the charge of the reactant, like iodoacetate and iodoacetamide [15]. These effects are attributed to the positive charge on the nicotinamide portion of NAD +, which provides additional positive charge at the active site of enzyme. Thus, the negative charge of koningic acid may contribute its high-affinity interaction with NAD÷-enzyme complex. Koningic acid has an epoxide moiety in the molecule (Fig. 1), which is known to be highly reactive. It has been shown that epoxides are attacked by nucleophiles in aqueous solution, and that sulfhydryl groups exhibit considerably greater nucleophilic reactivity toward epoxides than do amines, which in turn are more effective than oxygen nucleophiles [16,17]. These observations suggest that koningic acid binds to the sulfhydryl group of the enzyme. Glyceraldehyde-3-phosphate dehydrogenase displays the interesting properties of half-of-thesites reactivity with a variety of sulfhydryl reagents [18,19]. Thus, when two of the four activesite sulfhydryl groups are modified, the enzyme is catalytically inactive and the remaining two thiol groups have diminished reactivity toward the

modifiers. The stoichiometry of [3H]koningic acid binding to the enzyme and sulfhydryl group loss (Fig. 6) indicates complete loss of enzyme activity when two sulfhydryl groups are modified, possibly at the active site. These observations indicate that koningic acid is a half-of-the-sites inhibitor of the enzyme. But the detailed mechanism for the binding of koningic acid to sulfhydryl group of enzyme is to be studied further.

Acknowledgements The authors wish to thank Dr. S. Murakawa for valuable discussions, and Mrs. M. Takeda for typing the manuscript.

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