Roles of β-d -ribofuranose ring and the functional groups of the d -ribose moiety of adenosylcobalamin in the diol dehydratase reaction

Bioehimica et Biophysica Acta 952 (1988) 191-200 Elsevier

191

BBA 33052

Roles of the fl-D-ribofuranose ring and the functional groups of the D-ribose moiety of adenosylcobalamin in the dioi dehydratase reaction

Masakazu Ichikawa and Tetsuo Toraya Department of Chemistry, College of Liberal Arts and Sciences, Kyoto Unioersity, Kyoto (Japan) (Received 7 September 1987)

Key words: Adenosylcobalamin; Vitamin B-12 coenzyme; Diol dehydratase; Propanediol dehydratase; Carbon-cobalt bond activation; (K. pneumoniae)

Four analogs of adenosylcobalamin (AdoCbl) modified in the D-ribose moiety of the Coil ligand were synthesized, and their coenzymic properties were studied with diol dehydratase of Klebsiella pneumoniae ATCC 8724. 2'-Deoxyadenosylcobalamin (2'-dAdoCbl) and 3'-deoxyadenosylcobalamin (3'-dAdoCbl) were active as coenzyme. 2',3'-Secoadenosylcobalamin (2',3'-secoAdoCbi), an analog bearing the same functional groups as AdoCbl but nicked between the 2' and 3' positions in the ribose moiety, and its 2',3'-dialdehyde derivative (2',3'-secoAdoCbl dialdehyde) were totally inactive analogs of the coenzyme. It is therefore evident that the fl-D-ribofuranose ring itself, possibly its rigid structure, is essential and much more important than the functional groups of the ribose moiety for coenzymic function (relative importance: fl-D-ribofuranose ring >> 3'-OH > 2'-OH > ether group). With 2'-dAdoCbl and 3'-dAdoCbl as coenzymes, an absorption peak at 478 nm appeared during enzymatic reaction, suggesting homolysis of the C-Co bond to form cob(II)alamin as intermediate. In the absence of substrate, the complexes of the enzyme with these active analogs underwent rapid inactivation by oxygen. This suggests that their C-Co bond is activated even in the absence of substrate by binding to the apoprotein. No significant spectral changes were observed with 2',3'-secoAdoCbl upon binding to the apoenzyme. In contrast, spectroscopic observation indicates that 2',3'-secoAdoCbi dialdehyde, another inactive analog, underwent gradual and irreversible cleavage of the C-Co bond by interaction with the apodioi dehydratase, forming the enzyme-bound cob(II)alamin without intermediates.

Introduction An AdoCbl-dependent diol dehydratase (1,2propanediol hydro-lyase, EC 4.2.1.28) from Klebsiella pneumoniae is an enzyme which catalyzes the conversion of 1,2-propanediol, 1,2ethanediol and glycerol to propionaldehyde, acetaldehyde and /3-hydroxypropionaldehyde, re-

Correspondence: T. Toraya, Department of Chemistry, College of Liberal Arts and Sciences, Kyoto University, Sakyo-Ku, Kyoto 606, Japan.

spectively [1,2]. It is generally accepted that the C - C o bond of the coenzyme is labilized by binding to diol dehydratase and cleaved homolytically in the presence of substrate [3,4]. The enzymatic activation of the C-Co bond of the coenzyme is essential for manifestation of coenzymic function

Abbreviations: AdoCbl, adenosylcobalamin or Coa-[ a-(5,6-dimethylbenzimidazolyl)]-Cofl-adenosylcobamide; 2'-dAdoCbl, 2'-deoxyadenosylcobalamin; 3'-dAdoCbl, 3'-deoxyadenosylcobalamin; 2',3'-secoAdoCbl, 2',3'-secoadenosylcobalamin; 2',3'-secoAdoCbl dialdehyde, 2',3'-dialdehyde derivative of 2',3'-secoadenosylcobalamin.

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

192

in the AdoCbl-dependent enzyme reactions. During the course of our studies on the structure-function relationship of the coenzyme in the diol dehydratase system by using many AdoCbl analogs, it has been demonstrated that diol dehydratase possesses a specific binding site for the adenosyl group of the coenzyme [5,6], and that the interaction between this binding site in the enzyme and the adenosyl group of the coenzyme is essential for activation of the C-Co bond and thus for catalysis [5]. Importance of the nitrogen atoms in the adenine moiety of the coenzyme in the catalytic function has been evaluated by using deaza analogs of AdoCbl [7]. Recently, we have reported that adeninylethylcobalamin, an inactive analog, undergoes cleavage of the C-Co bond by interaction with apoprotein of diol dehydratase, although longer chain homologs do not [8]. This finding must be closely related with the role of the D-ribose moiety of the adenosyl group in catalysis and enzymatic activation of the C-Co bond. In order to investigate this problem further, coenzyme analogs modified in the ribose moiety of the Coil ligand were synthesized and tested for coenzymic activity in the diol dehydratase system. Our results are described here. Previously, the following ribose-modified analogs of AdoCbl have been synthesized and some properties reported: ara-adenosylcobalamin [9-11], aristeromycylcobalamin [9,11,12], 2'dAdoCbl [9,13-16], 3'-dAdoCbl [14], 2',3'-isopropylidene derivative of AdoCbl [9,15,17], L-adenosylcobalamin [9,11], homoadenosylcobalamin [10], adenosylethylcobalamin [9,11], 2'-(9-adeninyl)ethylcobalamin [8,9,11,18,19], 3'-(9-adeninyl)propylcobalamin [9,11,18,19], 4'-(9-adeninyl) butylcobalamin [9,11,18-21], 5'-(9-adeninyl) pentylcobalamin [9,11,18,19], and 6'-(9-adeninyl) hexylcobalamin [9,11,18,19]. Materials

and Methods

Materials Crystalline AdoCbl was a gift from Eisai Co., Tokyo, Japan. Cyanocobalamin was obtained from Glaxo Research Laboratories, Greenford, U.K. 2'-Deoxyadenosine and 3'-deoxyadenosine (cordycepin) were purchased from Kojin Co., Tokyo, Japan, and Yamasa shoyu Co., Chiba, Japan, re-

spectively. All other chemicals were reagent grade commercial products and were used without further purification•

Diol dehydratase The apoenzyme of diol dehydratase was purified as described before [22] from cells of Klebsiella pneumoniae ATCC 8724 (formerly Aerobacter aerogenes) grown without aeration in a glycerol/ 1,2-propanediol medium [23]. A highly purified preparation of the enzyme with specific activity more than 40 units/mg of protein was used throughout this study. The substrate-free apoenzyme was obtained by dialysis of apoenzyme overnight at 4 ° C against 1000 vol. of 0.05 M potassium phosphate buffer (pH 8.0). Enzyme and protein assays The activity of diol dehydratase was determined by the 3-methyl-2-benzothiazolinone hydrazone method [11] or the NADH-alcohol dehydrogenase coupled method [24]. 1 unit is defined as the amount of enzyme activity catalyzing the formation of 1/tmol of propionaldehyde per min under the standard assay conditions. For routine assays, the amount of enzyme to be assayed is kept between 0.001 and 0.05 unit [11,24]. The molar concentration of diol dehydratase was calculated on the basis of its molecular weight of 230000 [22,25]. Protein concentration was determined either by the method of Lowry et al. [26], or by measurement of the absorbance at 278 nm. An absorption coefficient of 5.27 for 10 mg of diol dehydratase per ml and for a 1 cm light path was used for the latter method [22]. Other analytical procedures The concentration of corrinoids was determined spectrophotometrically after converting them to dicyanocobalamin by reaction with 0.1 M KCN. Organocobalamins were converted into the dicyano form by photolysis in the presence of KCN. e367 for dicyanocobalamin is 30.4- 103 M-1 • cm -1 [27]. Visible and ultraviolet spectra were measured on a Union model SM-401 recording spectrophotometer. Thin-layer chromatography was performed on Merck silica gel G-60 glass plates. The solvent systems used are: (A) 1butanol/2-propanol/water (10 : 7 : 10, v/v); (B)

193 water-saturated 2-butanol; (C) water-saturated 2butanol containing 1% ( v / v ) acetic acid; (D) water-saturated 2-butanol containing 1% ( v / v ) (28%) N H 4 O H ; (E) ethyl a c e t a t e / m e t h a n o l (6:1, v / v ) . Paper electrophoresis in 0.5 M acetic acid (pH 2.7) was also carried out for analysis and purification of corrinoids.

HO OH

HO H

Ao

CH 2

CH2

2'-dAdoCbl

AdoCbl

HO OH

H OH

CH2

3'-dAdoCbl

?i 0

Synthesis of 5 '-O-tosyldeoxyribonucleosides 2'-Deoxy-5'-O-tosyladenosine was synthesized from 2'-deoxyadenosine by reaction with tosyl chloride (p-toluenesulfonyl chloride) in dry pyridine, as described by Zagalak et al. [14]. 3'-Deoxy5'-O-tosyladenosine was also prepared in the same way. R5,_o_tosyla d . . . . ine for 2'-deoxy and 3'-deoxy analogs of 5'-O-tosyladenosine in solvent E was 1.08 and 1.04, respectively.

2 ', 3 ' - seco Ado Cbl

2 ', 3'-seco AdoCb/ diatdehyde

Fig. 1. Ribose moieties of AdoCbl and the analogs used in this study. Ad, adenine; ~ , cobalamin.

Synthesis of ribose-modified analogs of AdoCbl The partial structures of the coenzyme analogs used in this study are summarized in Fig. 1. 2'dAdoCbl and 3'-dAdoCbl were synthesized from the corresponding 5'-O-tosyldeoxyribonucleosides by an established procedure [10,28]. Each 5 ' - 0 t o s y l d e o x y r i b o n u c l e o s i d e was reacted with cob(I)alamin which was formed from CN-Cbl by reduction with N a B H 4 or zinc p o w d e r / N H a C 1 . The reaction mixture was filtered and desalted by phenol extraction. The coenzyme analogs formed were purified by phosphocellulose (adjusted to p H 3 or 6) column chromatography and paper electrophoresis in 0.5 M acetic acid (pH 2.7). To confirm

the structures, 2'-dAdoCbl and 3'-dAdoCbl were photolyzed under aerobic conditions. The products derived from the Coil ligands were reduced with NaBH4, and then analyzed by thin-layer chromatography on silica gel. As expected, 2'-deoxyadenosine and 3'-deoxyadenosine were formed from 2'-dAdoCbl and 3'-dAdoCbl, respectively. 2',3'-SecoAdoCbl and its 2',3'-dialdehyde derivative were prepared as described below. AdoCbl was treated with 0.01 M K I O 4 in 0.01 M acetic acid for 2 h at room temperature. Excess IO 4 was destroyed by adding 1,2-propanediol to the reac-

TABLE I CHROMATOGRAPHIC AND ELECTROPHORETIC BEHAVIORS OF COENZYME ANALOGS Relative mobility in paper electrophoresis at pH 2.7 b

Cobalamin

R ¢N-Cbl in thin-layer chromatography a Solv. A Solv. B Solv. C

Solv. D

AdoCbl 2'-dAdoCbl 3'-dAdoCbl 2',3'-secoAdoCbl (Analog II) 2',3'-secoAdoCbl dialdehyde (Analog I) Aquacobalamin

0.79 0.81 0.92 0.79

0.83 0.86 1.03 0.83

0.72 0.83 0.90 0.76

0.76 0.81 0.65 0.76

1.17 1.19 1.03 1.03

0.81 0.10

0.79 0.20

0.76 0.39

0.69 0.04

0.73 ( -=1)

a On Merck silica gel G-60 precoated plates. b In 0.5 M acetic acid (pH 2.7) at a voltage gradient of about 22 V/cm. Mobility of cyanocobalamin and aquacobalamin was taken as 0 and 1, respectively.

194 TABLE II ABSORPTION SPECTRA OF RIBOSE-MODIFIED ANALOGS Cobalamin In water 2'-dAdoCbl 3'-dAdoCbl 2',3'-secoAdoCbl (Analog II) 2',3'-secoAdoCbl dialdehyde (Analog 1) In 0.1 M HCI 2'-dAdoCbl 3'-dAdoCbl 2',3'-secoAdoCbl (Analog II) 2',3'-secAdoCbl dialdehyde (Analog I)

~'m,,~(nm)(e×10 3, M-l.cm a) 262 (36.4) 262 (37.5) 260 (35.3) 259 (39.6)

278s ~ (23.1) 280s (23.8) 280s (22.0) 278s (24.8)

317 (13.3) 316 (13.9) 320 (12.6) 320 (12.9)

339 (13.1) 340 (13.9) 338 (13.6) 338 (13.9)

377 (11.2) 376 (11.6) 376 (10.2) 375 (12.0)

435 (4.4) 437 (4.8) 439 (4.3) 432 (4.5)

526 (8.6) 526 (8.9) 526 (9.0) 528 (9.0)

263 (45.6) 263 (44.0) 264 (38.5) 262 (42.9)

276s (30.1) 274s (30.1) 275s (32.1) 277s (28.1)

289 (26.1) 285 (25.3) 284 (23.8) 284 (24.9)

304 (24.9) 303 (24.2) 304 (19.1) 304 (20.0)

315s (22.3) 315s (21.5) 314s (17.9) 314s (19.6)

380 (9.2) 380 (8.9) 374s (7.3) 373 (8.0)

460 (10.1) 460 (9.5) 456 (8.2) 459 (9.2)

" s, shoulder.

tion mixture. The reaction mixture was then desalted by phenol extraction. After passing through a phosphocellulose (adjusted to p H 6) column to remove a trace of aquacobalamin, the products were subjected to paper electrophoresis in 0.5 M acetic acid (pH 2.7). An organocobalamin migrating more slowly than AdoCbl (Table I) was the major product which we designated Analog I. Analog I was freed from oxidized (carboxyl-group containing) impurities by passing through a DEAE-cellulose (acetate form) column. The yield of Analog I was approx. 80 90%. Analog I was reduced with N a B H 4 at 0 ° C in 0.1 M potassium phosphate buffer (pH 8.0-8.4). After desalting by phenol extraction, the corrinoid products formed were passed through a phosphocellulose (pH 6) column to remove aquacobalamin, a major byproduct, and then subjected to paper electrophoresis in 0.5 M acetic acid. A new organocobalamin which moved as fast as AdoCbl was designated Analog II. Analog II was separated from Analog I by paper electrophoresis at pH 2.7. The analogs thus obtained were chromatographically pure in four solvent systems listed in Table I. Relative mobility in paper electrophoresis at pH 2.7 is also shown in Table I. The absorption

spectra of these analogs measured in neutral and acidic solutions are summarized in Table II. Structural identification of Analogs II and I as 2',3'-secoAdoCbl and its 2',3'-dialdehyde derivative, respectively, is described in the next section. Results

Identification of Analogs I and H To elucidate the structures of Analogs I and II, their C - C o bond was cleaved by reduction with H 2 / P t O 2, and the degradation products derived from the Coil ligands were compared with authentic compounds by thin-layer chromatography on silica gel. As expected, the nucleoside products from Analogs II and I coincided with 5'-deoxy-2',3'-secoadenosine and its 2',3'-dialdehyde derivative, respectively (R F in solvent E, 0.16 and 0.38, respectively). The product of aerobic photolysis of Analog I was converted to 2',3'-secoadenosine by reduction with N a B H 4. These results indicate that Analogs II and I are 2',3'-secoAdoCbl and its 2',3'-dialdehyde derivative (2',3'-secoAdoCbl dialdehyde), respectively.

Chemical properties of AdoCbl analogs As illustrated in Fig. 2, 2',3'-secoAdoCbl dial-

195 B

10£

/

5c

3'-dAdoCbl x

-0,5

\

2'- d A d o C b l \ \

\

x

O -1 0 ~

.< <3

u

AdoCbl

-15 0

6 ~ r U

X

X

X

120 0 Time (min)

60

0

120

ib

~o

Time (min)

Fig. 2. Chemical stability of 2',3'-secoAdoCbl dialdehyde (A) and AdoCbl (B). The analog or AdoCbl (I0 ~M) was treated in the dark for the indicated time periods under the following conditions, and the extent of the C-Co bond cleavage was determined spectrophotometrically. ©, in 1.0 M NH4OH at 37°C; zx, in 0.1 M HC1 at 100°C; x, in0.05 M potassium phosphate buffer (pH 8.0) at 37 o C. dehyde was m u c h more labile to 1 M N H 4 O H t h a n A d o C b l , although it was quite stable in p o t a s s i u m p h o s p h a t e buffer (pH 8.0). Toward 0.1 M HC1, however, this analog was more stable than A d o C b l . T h e other three analogs were as stable as A d o C b l in the alkaline a n d acidic solutions (data n o t shown). As shown in T a b l e I, relative mobility of 2 ' , 3 ' - s e c o A d o C b l dialdehyde in paper electrophoresis at p H 2.7 was m u c h smaller than that of A d o C b l or the other three analogs. F u r t h e r m o r e , the p K a value of 2.7 for this analog in the b a s e - o n

Fig. 3. Time-course of diol dehydratase reaction with AdoCbl, 2'-dAdoCbl and 3'-dAdoCbl as coenzymes. The NADH-alcohol dehydrogenasecoupled assay method was employed. The reaction mixture was composed of 0.03 unit of apoenzyme, 0.2 M 1,2-propanediol, 50 ,ttg of yeast alcohol dehydrogenase, 0.3 mM NADH, 0.04 M potassium phosphate buffer (pH 8.0), and 15/~M AdoCbl or its analog in a total volume of 1.0 ml. The reaction started by addition of coenzyme was carried out at 37°C. base-off e q u i l i b r i u m was m u c h lower than that for A d o C b l ( p K , 3.5) [29]. F r o m these results, it seems evident that the cobalt atom in 2',3'-seco A d o C b l dialdehyde is more electrophilic than that in A d o C b l or the other three analogs.

Coenzymic activity of AdoCbl analogs in diol dehydratase system T a b l e III summarizes the kinetic properties of the coenzyme analogs. Of the analogs examined, 2 ' - d A d o C b l a n d 3 ' - d A d o C b l were partially active

TABLE llI KINETIC CONSTANTS FOR AdoCbl AND ITS ANALOGS IN THE DIOL DEHYDRATASE REACTION

Cobalamin

kcat a (s- 1)(%)

kinact b (min -1 )

(kcat/kinact) × 10 -4

Km (~tM)

AdoCbl 2"-dAdoCbl Y-dAdoCbl 2',3'-secoAdoCbl 2',3'-secoAdoCbl dialdehyde

337 c (100) 103 (31) 63 (19) inactive inactive

0.014 d 0.112 0.051

144 d 5.5 7.4

0.80 e 1.3 2.3

Ki (ttM)

4.0 5.8

a Calculated from the maximum velocity. b Calculated from a change in the slope of a tangent to the time-course curve of the reaction with 15 ~M each coenzyme, which was determined by the NADH-alcohol dehydrogenase-coupled method [24]. c From Ref. 2. d From Ref. 24. c From Ref. 11.

196 coenzymes in the diol dehydratase system (relative coenzyme activity calculated from Vm~x, 31 and 19%, respectively). As shown in Fig. 3, the time-course of the 1,2-propanediol-dehydration reaction with 2'-dAdoCbl or 3'-dAdoCbl as coenzyme deviated from linearity. This and the kcat/ kinact values in Table III indicate that both the complexes of enzyme with 2'-dAdoCbl and 3'dAdoCbl gradually lose activity during catalysis, although the inactivation rate with the former is faster than that with the latter. As judged from the apparent K m values, the affinity of the enzyme for 2'-dAdoCbl was somewhat higher than that for 3'-dAdoCbl. 2',3'-SecoAdoCbl and its 2',3'-dialdehyde analog were quite inactive as coenzyrnes. These inactive analogs served as competitive inhibitors with respect to AdoCbl. From their ap-

A

Free 2'-dAdoB12

parent K i values, the affinity of the enzyme for these analogs is still fairly high. Since 2',3'-secoAdoCbl bears the same functional groups as AdoCbl in the ribose moiety, it can be concluded that the contribution of the fl-D-ribofuranose ring to coenzymic function is essential and much greater than those of the functional groups in the ribose moiety.

Spectroscopic study The extent of activation and cleavage of the C - C o bond by the binding to apoprotein was estimated by comparing the spectra of enzymeanalog complexes in the presence of substrate with those of the corresponding free analogs. As shown in Figs. 4B and D, a new peak appeared at 478 nm in the spectra of the complexes of enzyme with

C

Free 3"-dAdoBi2

0.05 <

!

I

B / 60: 120' ~/j 30"

0.05

apoE E' 2'-dAdo812

~00',

........

D

.....

apoE E 3'-dAdoO12

........

hv (after 2hr)

hv(after 2 hr)

O" <

"~-. ~ 0 " . ,._~.

O'

120"

10'

400

500 Wavelength

600 (rim)

400

500 Wavelength (nm)

600

Fig. 4. Optical spectra of active coenzymeanalog-enzymecomplexes in the presence of substrate. Apoenzyme(40 units, 2.5 nmol) was incubated with 2.0 nmol of each coenzyme analog at 37°C in the presence of 0.2 M 1,2-propanediol and 0.04 M potassium phosphate buffer (pH 8.0) in a total volume of 1.0 ml. Spectra were taken at the time indicated. Spectra of the apoenzyme(bottom spectra in B and D) and each free coenzymeanalog at the same concentration were measured as controls. Photolysis was carried out at 0 °C for 10 min with a 200 W tungsten light bulb at a distance of 10 cm. A, free 2'-dAdoCbl; B, enzyme-2'-dAdoCblcomplex; C, free 3'-dAdoCbl; D, enzyme-3'-dAdoCblcomplex.

197

2'-dAdoCbl and 3'-dAdoCbl in the presence of 1,2-propanediol (reacting complexes), suggesting formation of cob(II)alamin as intermediate. This indicates that homolysis of the C-Co bond takes place during catalysis with these analogs as well as with regular coenzyme (AdoCbl). In the case of the enzyme-2'-dAdoCbl complex (Fig. 4B), the intensity of the peak at 478 nm reached maximum within about 10 min and then decreased gradually with time of incubation. In addition, the absorption peak at 375 nm decreased and a newly appeared peak at 356 nm increased with time and reached maximum within 2 h. By this time of incubation the enzyme-2'-dAdoCbl complex was completely inactivated. No spectral changes were observed anymore upon photolysis at 2 h, indicating complete and irreversible cleavage of the C - C o bond of the enzyme-bound analog by this time. Therefore it is very likely that the gradual loss of enzyme activity is related to the slow and irreversi-

ble conversion of 2'-dAdoCbl to the enzyme-bound hydroxocobalamin during catalysis at 37°C. As compared with this, the spectral changes observed with the enzyme-3'-dAdoCbl complex upon prolonged incubation were rather inconspicuous (Fig. 4D). This complex was also inactivated completely within 2 h, and the spectrum obtained finally was similar to that of enzyme-bound cob(II)alamin [5]. Again, no spectral changes were observed upon photolysis at 2 h, indicating complete and irreversiblecleavage of the C-Co bond of this analog. Thus, it seems probable that enzyme-bound 3'dAdoCbl is irreversibly converted to cob(II) alamin, accompanied with slow inactivation of the enzyme-3'-dAdoCbl complex. Spectral changes of inactive analogs upon binding to the apoenzyme are illustrated in Fig. 5. No appreciable spectral differences between free and the enzyme-bound 2',3'-secoAdoCbl were observed even in the presence of substrate (Fig.

Free 2', 3'-secoAdoB12

A .........

C

Free 2'.3'-seco AdoB12 d i aldehyde

hv

.......... hv

0.05 ,/'\

<

I

. . . . . . . . apoE - - . E- 2',3'-secoAdoB12 ........... h ~ ( a f t e r 2 hr)

B 0.05

D ~i~r~. ~" i

.

i

......

apoe

,--

E'Z',3'-Sec°Ad°BI2

I i~

~

"~

i. . . . . . .

dialdehyde h v ( a f t e r 3 hr)

i 0". I0". 20', 30'. 60'.120" --.

400

500

600

400

500

600

W a v e l e n g t h ( nm ) Wavelength (nm) Fig. 5. Optical spectra of inactive coenzyme analog-enzyme complexes in the presence of substrate. The experimental conditions are the same as those described in Fig. 4. A, free 2',3'-secoAdoCbl; B, enzyme-2',3'-secoAdoCbl complex; C, free 2',3'-secoAdoCbl dialdehyde; D, enzyme-2',3'-secoAdoCbl dialdehyde complex. Inset, A 0, A t and A~ are absorbance at 525 nm at the time points of 0, t and 180 min, respectively.

198

5A and B). It is therefore clear that the C-Co bond of 2',3'-secoAdoCbl was not cleaved by the binding of the analog to the enzyme. As comapred with the free counterpart, the enzyme-bound 2',3'-secoAdoCbl was highly resistant to photolysis (Fig. 5B). This may be due to rapid recombination of cob(II)alamin and the CoB ligand-derived organic radical both of which are retained at the active site [11]. In contrast, the spectrum of the enzyme-2',3'-secoAdoCbl dialdehyde complex changed definitely with time of incubation (Fig. 5C and D): the absorption peaks at 375 nm and 525 nm decreased and a new absorption peak at 478 nm increased with clear isosbestic points. The spectrum obtained at 3 h no longer changed upon photo-irradiation, and was very similar to that of enzyme-bound cob(II)alamin [5]. Thus, it is clear that the C-Co bond of the analog was completely cleaved by this time, forming cob(II)alamin without intermediates. By taking the absorbance at 3 h as Am, the time-course of the C-Co bond cleavage can be followed from the decrease of absorbance at 525 nm. As shown in the inset of Fig. 5D, a plot of logarithm of the percentage of 2',3'-secoAdoCbl dialdehyde remaining against the incubation time was linear after a short lag period (about 10 min, probably required for the binding step), indicating that the conversion of the analog to the enzyme-bound cob(II)alamin obeyed firstorder reaction kinetics with a first-order rate constant (k) of 0.024 min 1 (tl/2 = 29 rain) at 37°C. Since this analog itself was quite stable in 0.05 M potassium phosphate buffer (pH 8.0) at 37 ° C (the conditions for diol dehydratase reaction), these results indicate that 2',3'-secoAdoCbl dialdehyde undergoes activation and homolysis of its C-Co bond by interaction with the apodiol dehydratase, although it does not function as coenzyme. The cob(II)alamin formed from the enzyme-bound 2',3'-secoAdoCbl dialdehyde was stable for at least 2 h even in the presence of atmospheric oxygen.

02-Inactwation of the enzyme-analog complexes in the absence of substrate Aerobic incubation of diol dehydratase apoenzyme with AdoCbl in the absence of substrate leads to total inactivation of enzyme accompanying irreversible cleavage of the C-Co bond of the enzyme-bound AdoCbl [1,30]. This is believed to

10(~

i

..........

x--

50

Preincubotion time (min)

Fig. 6. Inactivation of enzyme-AdoCbl and enzyme-coenzyme analog complexes in the absence of substrate. The substrate-free apoenzyme (0.16 unit) was aerobically incubated at 37 °C with and without 15 ffM AdoCbl or analog in 0.9 ml of 0.04 M potassium phosphate buffer (pH 8.0). After the indicated time of incubation, 0.2 mmol of 1,2-propanediol was added to a final volume of 1.0 ml, and the remaining activity was measured by 10 min incubation at 37°C. x , apoenzyme; e, enzyme-AdoCbl complex; O, enzyme-2'-dAdoCbl complex; zx, enzyme-3'-dAdoCbl complex.

be due to the reaction of the activated C-Co bond of the coenzyme with oxygen. When 2'-dAdoCbl or 3'-dAdoCbl was incubated with the apoenzyme, the resulting complex underwent rapid inactivation by oxygen, as shown in Fig. 6. Therefore, it can be concluded that these analogs also undergo activation of the C-Co bond by the binding to apoprotein even in the absence of substrate. Discussion

It was previously reported from this and other laboratories that some of the analogs of AdoCbl modified in the D-ribose moiety of the Coil ligand, such as ara-adenosylcobalamin, aristeromycylcobalamin, and 2'-dAdoCbl retained partial coenzymic activity in the diol dehydratase system [11-13]. The analogs in which the ribose moiety are replaced by L-ribose, D-ribosylethyl, or alkyl groups, such as L-adenosylcobalamin, adenosylethyl-cobalamin, 2'-(9-adeninyl)ethylcobalamin, 3'-(9-adeninyl)propylcobalamin, 4'-(9-adeninyl) butylcobalamin, 5 '-(9-adeninyl)pentylcobalamin, 6'-(9-adeninyl)hexylcobalamin, have been reported to be inactive analogs [8,11]. In the present study, the coenzyme analogs modified in the D-

199 ribose moiety of the adenosyl group were synthesized to estimate the relative importance of the /3-o-ribofuranose ring and the functional groups attached to it in the coenzymic function. 2'dAdoCbl and 3'-dAdoCbl were 31 and 19% as active as regular coenzyme in the diol dehydratase reaction. Hogenkamp and Oikawa [13] have also reported that 2'-dAdoCbl is a partially active coenzyme in this system. These data, together with the fact that aristeromycylcobalamin, a carbocyclic analog, is 42% as active as AdoCbl [11,12], indicate that the functional groups of the ribose moiety, i.e., 2'- and 3'-hydroxyl groups and the ether group, are important, but not absolutely required for catalytic action of the coenzyme. 2',3'-SecoAdoCbl, an analog which bears the same functional groups as AdoCbl but is nicked between the 2' and 3' positions, was totally inactive as coenzyme. It is therefore clear that the /3-0ribofuranose ring itself is essential and much more important than the functional groups of the ribose moiety for manifestation of coenzymic function (relative importance decreasing in the following order: /3-D-ribofuranose ring >> 3'-OH > 2'-OH > -O-). It is noteworthy that both the complexes of the enzyme with 2'-dAdoCbl and 3'-dAdoCbl gradually become inactive during catalysis. The same phenomenon was observed in common with other ribose-modified active analogs, e.g., ara-adenosylcobalamin and aristeromycylcobalamin [11]. In contrast, the complexes of the enzyme with adenine-modified active analogs do not undergo significant inactivation i~ the course of catalysis [7,11,31]. Toraya and Abeles [32] have proposed that inactivation of diol dehydratase by AdoCbl e-carboxylic acid during catalysis is due to incorrect binding of the modified coenzyme in an intermediate of the catalytic process which lead to the loss of substrate radical. Therefore, proper interaction of the enzyme with AdoCbl at the D-ribose moiety of the Co/3 ligand may contribute to organization of the very rigid intermediate complexes. 2',3'-SecoAdoCbl did not undergo activation of the C-Co bond by diol dehydratase, although it contains not only the adenine ring but also the same functional groups as AdoCbl in the ribose moiety. In the preceding paper [8], we proposed

that the strong interaction between the adenine moiety of the coenzyme and the adenosyl groupbinding site of the enzyme produces a kind of tensile force or angular strain between the adenine and the cobalt atom, which is at least one element of the force that weakens the C-Co bod of the coenzyme or 2'-(9-adeninyl)ethylcobalamin. In the case of 2',3'-secoAdoCbl, in analogy with 4'-(9adeninyl)butylcobalamin, such strain or force must be relieved by a flexible chain which combines the adenine ring with the cobalt atom. This would be a possible reason why the C-Co bond of 2',3'-secoAdoCbl was not activated by the enzyme. It seems thus very likely that the rigid structure of the fl-o-ribofuranose ring is important to transmit the strain or force to the C-Co bond. On the contrary, the C-Co bond of 2',3'-secoAdoCbl dialdehyde underwent gradual cleavage by the enzyme forming the enzyme-bound cob(II)alamin without intermediates. This is the second coenzymically inactive analog whose C-Co bond undergoes cleavage by diol dehydratase. The results demonstrate that the ribose moiety is essential for coenzymic activity but not absolutely necessary for labilization of the C-Co bond by the enzyme. Spectral observation of cob(II)alamin shows that the cleavage of the C - C o bond is by homolysis, although the product derived from the Coil ligand is not yet identified. Hence, this is in clear contrast to 2'-(9-adeninyl)ethylcobalamin. The latter is the cobalamin with which cleavae of the C-Co bond of an inactive analog by diol dehydratase has been first demonstrated [8], but its C - C o bond cleavage seems to be by heterolysis forming hydroxocobalamin. Since an early event in the widely accepted mechanism for diol dehydratase reaction is the homolytic cleavage of the C - C o bond of the coenzyme [3,4], cleavage of the C - C o bond of 2',3'-secoAdoCbl dialdehyde by the interaction with the apoenzyme is interesting in relation with the C-Co bond homolysis of AdoCbl during catalysis. This peculiarity of 2',3'secoAdoCbl dialdehyde shown in the enzymatic C - C o bond activation seems owing to the intrinsic chemical reactivity of its C-Co bond. It is much more sensitive to alkali but more stable to acid than the other analogs or AdoCbl itself. From its lower mobility in paper electrophoresis at pH 2.7 and its lower pK a value in the base-on

200

base-off equilibrium, the cobalt atom in this analog is supposed to be more electrophilic than that in AdoCbl or the other analogs. It is very likely that these unique properties result from introduction of the aldehyde group into the /3 position. Thus, a possibility can be considered that activation and cleavage of the C-Co bond of this analog by the enzyme is not owing to the specific interaction at the adenine moiety but due to the intrinsic instability of its C-Co bond. This possibility is under current investigation. References 1 Lee, H.A., Jr. and Abeles, R.H. (1963) J. Biol. Chem. 238, 2367-2373. 2 Toraya, T., Shirakashi, T., Kosuga, T. and Fukui, S. (1976) Biochem. Biophys. Res. Commun. 69, 475-480. 3 Abeles, R.H. and Dolphin, D. (1976) Acc. Chem. Res. 9, 114-120. 4 Toraya, T. and Fukui, S. (1982) in B]2 (Dolphin, D., ed.), Vol. 2, pp. 233-262, John Wiley & Sons, New York. 5 Toraya, T. (1985) Arch. Biochem. Biophys. 242, 470-477. 6 Toraya, T., Watanabe, N., Ushio, K., Matsumoto, T. and Fukui, S. (1983) J. Biol. Chem. 258, 9296-9301. 7 Toraya, T., Matsumoto, T., Ichikawa, M., Itoh, T., Sugawara, T. and Mizuno, Y. (1986) J. Biol. Chem. 261, 9289-9293. 8 Toraya, T., Watanabe, N., Ichikawa, M., Matsumoto, T., Ushio, K. and Fukui, S. (1987) J. Biol. Chem. 262, 8544-8550. 9 Sando, G.N., Blakley, R.L., Hogenkamp, H.P.C. and Hoffmann, PJ. (1975) J. Biol. Chem. 250, 8774-8779. 10 Jacobsen, D.W., DiGirolamo, P.M. and Huennekens, F.M. (1975) Mol. Pharmacol. 11, 174-184. 11 Toraya, T., Ushio, K., Fukui, S. and Hogenkamp, H.P.C. (1977) J. Biol. Chem. 252, 963-970.

12 Kerwar, S.S., Smith, T.A. and Abeles, R.H. (1970) J. Biol. Chem. 245, 1169-1174. 13 Hogenkamp, H.P.C. and Oikawa, T.G. (1964) J. Biol. Chem. 239, 1911-1916. 14 Zagalak, B. and Pawelkiewicz, J. (1965) Acta Biochem. PoL 12, 219-228. 15 Vitols, E., Brownson, C., Gardiner, W. and Blakley, R.L (1967) J. Biol. Chem. 242, 3035-3041. 16 Babior, B.M. (1969) J. Biol. Chem. 244, 2917-2926. 17 Pawelkiewicz, J. and Zagalak, B. (1964) Ann. NY Acad. Sci. 112, 703-705. 18 Sando, G.N., Grant, M.E. and Hogenkamp, H.P.C. (1976) Biochim. Biophys. Acta 428, 228-232. 19 Krouwer, J.S., Holmquist, B., Kipnes, R.S. and Babior, B.M. (1980) Biochim. Biophys. Acta 612, 153-159. 20 Babior, B.M. (1969) J. Biol. Chem. 244, 2917-2926. 21 Ushio, K., Honda, S., Toraya, T. and Fukui, S. (1982) J. Nutr. Sci. Vitaminol. 28, 225-236. 22 Poznanskaja, A.A., Tanizawa, K., Soda, K., Toraya, T. and Fukui, S. (1979) Arch. Biochem. Biophys. 194, 379-386. 23 Abeles, R.H. (1966) Methods Enzymol. 9, 686-689. 24 Toraya, T., Krodel, E., Mildvan, A.S. and Abeles, R.H. (1979) Biochemistry 18, 417-426. 25 Essenberg, M.K., Frey, P.A. and Abeles, R.H. (1971) J. Am. Chem. Soc. 93, 1242-1251. 26 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 27 Barker, H.A., Smyth, R.D., Weissbach, H., Munch-Peterson, A., Toohey, J.I., Ladd, J.N., Volcani, B.E. and Wilson, M.R. (1960) J. Biol. Chem. 235, 181-190. 28 Hogenkamp, H.P.C. (1974) Biochemistry 13, 2736-2740. 29 Ladd, J.N., Hogenkamp, H.P.C. and Barker, H.A. (1961) J. Biol. Chem. 236, 2114-2118. 30 Wagner, O.W., Lee, H.A., Jr., Frey, P.A. and Abeles, R.H. (1966) J. Biol. Chem. 241, 1751-1762. 31 Ushio, K., Fukui, S. and Toraya, T. (1984) Biochim. Biophys. Acta 788, 318-326. 32 Toraya, T. and Abeles, R.H. (1980) Arch. Biochem. Biophys. 203, 174-180.