228
Biochimica et Biophysica Acta, 428 (1976) 228--232
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27829
THE INTERACTION OF ADENINYLALKYLCOBALAMINS WITH RIBONUCLEOTIDE REDUCTASE
GLORIA N. SANDO, MICHAEL E. GRANT and HARRY P.C. HOGENKAMP Department of Biochemistry, College of Medicine, University of Iowa, Iowa City, Iowa 52242 (U.S.A.)
(Received August 26th, 1975)
Summary Several structural analogs of adenosylcobalamin, containing 2, 3, 4, 5 and 6 methylene carbons instead of the ribofuranose moiety, have been synthesized and their interaction with ribonucleotide reductase from Lactobacillus leichmannii has been investigated. Kinetic studies of the inhibition of the reductase by these analogs showed that the adeninylalkylcobalamins with 4, 5 and 6 carbons interposed between the adenine moiety and the cobalt atom are potent inhibitors of ribonucleotide reduction. The stronger interaction between adeninylpentylcobalamin and the enzyme than that between adenosylcobalamin and the enzyme suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the protein upon binding.
Introduction Ribonucleotide reductase catalyzes the irreversible reduction of ribonucleotides to 2'-deoxyribonucleotides. The reductases from several microorganisms, including that of Lactobacillus leichmannii, have an absolute requirement for adenosylcobalamin as a coenzyme [1]. Recently we have examined a series of analogs of adenosylcobalamin f o r their effects on reactions catalyzed by ribonucleotide reductase of L. leichmannii [2]. The results of that study indicated that relatively small changes in either the nucleoside base or the sugar moiety attached to cobalt in the sixth coordination position profoundly affect coenzymic activity. One of these analogs, adenosylethylcobalamin, with two extra methylene groups interposed between the 5'-deoxyadenosine moiety and the cobalt atom was a very potent inhibitor of ribonucleotide reduction. Analogs in which the ribose moiety was replaced by three or four methylene carbons were inhibitory to a lesser extent, suggesting an effect of the carbon chain-length on the ability to interact with the enzyme. These results led us to synthesize a series of analogs of adenosylcobalamin in which the ribofuranose
229 moiety is replaced by 2, 3, 4, 5 and 6 methylene carbons. This communication describes these syntheses as well as the action of the analogs as inhibitors of ATP reduction by the ribonucleotide reductase of L. leichmannii. Materials and Methods
Materials. Cyanocobalamin and 1-bromo-5-chloropentane were obtained from Sigma Chemical Co.; 1-bromo-2-chloroethane, 1-bromo-3-chloropropane, 1-bromo-4-chlorobutane and 1-bromo-6-chlorohexane were supplied by Aldrich and J.T. Baker Chemical Co. Ribonucleotide reductase from L. leichmannii was kindly supplied by Dr. R.L. Blakley of the University of Iowa. [5'-3H~ ] Adenosylcobalamin (22 Ci/mol) and adenosylcobalamin were prepared as described before [ 3,4]. The 9-(w-chloroalkyl)adenines and adeninylalkylcobalamins were prepared by published procedures [5,6]. Methods. Ultraviolet and visible spectra were recorded on a Cary Model 15 spectrophotometer. Other absorbance measurements were made with a Zeiss PM Q II spectrophotometer. Ionization constants were determined by the spectral method of Ladd et al. [7]. Molar extinction coefficients of the cobalamins are based on e36 s = 30.8" 103 M -1 • cm -I for dicyanocobalamin [8]. The cobalamins were converted to dicyanocobalamin by photolysis in the presence of 0.1 M KCN. Descending chromatography on Whatman No. 1 paper was conducted with the following solvent systems: solvent I, sec-butylalcohol/ water/NH4OH (50 : 36 : 14, v/v); II, 1-butanol/ethanol/water (50 : 15 : 35, v/v); III, 1-butanol/isopropyl alcohol/water (37 : 26 : 37, v/v). Radioactivity measurements were made with a Packard Model 2420 TriCarb liquid scintillation spectrometer [9]. Assay procedures. The ability of the adeninylalkylcobalamins to function as coenzymes or inhibitors in the ribonucleotide reductase reaction was determined by two methods. Ribonucleotide reductase activity was determined by measuring the a m o u n t of dATP formed from ATP by the diphenylamine procedure of Blakley and co-workers [10,11]. Reaction mixtures contained 0.5 M potassium phosphate (pH 7.5), 30 mM dithiothreitol, 10 mM ATP, adenosylcobalamin and/or adeninylalkylcobalamin and ribonucleotide reductase (25 pg with a specific activity of approx. 150 units/mg) in a 0.5 ml volume. The reaction was initiated by the addition of all other components to a solution of the cobalamin. After a 10-min incubation of the reaction mixtures in the dark at 37°C, the tubes were placed in ice and the a m o u n t of dATP formed determined [2]. Ribonucleotide reductase activity was also determined by measuring tritium exchange between [ 5'- 3H2 ] adenosylcobalamin and water [ 12]. The reaction mixtures were identical to those described above, except that labeled adenosylcobalamin and less ribonucleotide reductase (0.4--0.8 pg) was used. Steady-state kinetic constants were determined as described before [2]. Results and Discussion
Physical and chemical properties of the adeninylalkyl cobalamins The adeninylalkylcobalamins containing 2, 3, 4 and 5 methylene groups are readily crystallized from 90% aqueous acetone, however, adeninylhexylcobala-
23O TABLE I PAPER CHROMATOGRAPHIC
PROPERTIES OF ADENINYLALKYLCOBALAMINS
N u m b e r of m e t h y l e n e
R ( c y a n o c o b a l a m i n values in i n d i c a t e d s o l v e n t s )
groups
Solvent I
S o l v e n t II
S o l v e n t III
2 3 4 5 6
1.10 0.63 0.76 1.28 1.90
1.03 0.78 0.98 1.15 1.26
1.07 0.93 0.97 1.18 1.28
min is quite soluble in acetone and was crystallized from a concentrated aqueous solution. All crystalline preparations were chromatographically homogeneous in solvents I, II and III (Table I). The absorption spectra of the adeninylalkylcobalamins are presented in Table II. The spectra of the cobalamins with two and three methylene groups interposed between the adenine moiety and the cobalt atom are very similar to that of adenosylcobalamin with a bands at 524 and 522 nm, respectively. In contrast the spectra above 400 nm of the cobalamins containing 4, 5 and 6 methylene groups resemble that of ethylcobalamin with broad maxima at 507, 515, and 509 nm. As in the case of adenosylcobalamin, acidification of aqueous solutions of the adeninylalkytcobalamins causes a spectral shift to lower wavelength. The pKa values estimated from the midpoints of these spectral changes reflect the positive inductive effect of the methylene groups (Table II). Thus the pK a of adeninylethylcobalamin {2.7) is very similar to that of methylcobalamin, while the PKa of the highest homologue, adeninylhexylcobalamin, is similar to that of ethylcobalamin (3.9) [13]. The stability of the carbon-cobalt bond of the adeninylalkylcobalamins is like that of the simple alkylcobalamins. Aqueous solutions of the adeninylalkylcobalamins are readily photolyzed especially in the pres-
T A B L E II ABSORPTION SPECTRA OF ADENOSYLCOBALAMIN
AND ADENINYL ALKYLCOBALAMINS
P o s i t i o n o f m a i n a b s o r p t i o n b a n d is given in n m . M o l a r e x t i n c t i o n c o e f f i c i e n t s (X 1 0 - 3 ) are given in parentheses.
N a t u r e of the sixth l i g a n d AdenosylAdeninyl Adeninyl Adeninyl Adeninyl Adeninyl
0.1 M HC1
266 (41.1) ethyl265 (43.7) propyl- 266 (40.8) butyl- 264 (38.8) pentyl- 265 (41.4) hexyl- 265 (37.3)
287 (23.9) 285 (24.6) 287 (23.5) 287 (22.1) 286 (23.0) 286 (21.3)
306 380 458 (22.2) (8.8)(9.4) 305 315 460 (19.9) (19.9) (9.9) 306 380 460 (23.8) (9.2) (9.6) 304 380 455 (25.7) (9.7) (9.0) 305 382 460 (27.3) (8.8) (8.4) 305 381 464 (24.9) (7.7) (7.9)
0.1 M p h o s p h a t e b u f f e r ( p H 7.0)
pK a
262 (35.1) 263 (38.1) 266 (34.4) 266 (31.8) 265 (33.0) 265 (33.4)
3.5
290 (18.2) 290 (18.7) 290 (18.6) 292 (17.5) 291 (17.6) 290 (18.9)
318 (13.0) 320 (12.4) 319 (13.9) 319 (14.4) 317 (14.5) 305 (14.8)
341 376 522 (12.8) (11.0) (8.0) 340 375 524 (13.4) (11.4) (8.9) 340 377 522 (13.2) (11.1) (8.2) 346 380 507 (12.7) (9.6) (8.3) 345 380 515 ( 1 2 . 5 ) (9.1) ( 9 . 4 ) 345 380 509 (11.6) (7.9) (7.9)
2.7 3.2 3.6 3.7 3.9
231
ence of oxygen, but they are not decomposed by acid (0.1 M HC1 at 90°C for 1 h) or by 0.1 M KCN.
Coenzyme activity of the adeninylalkylcobalamins As expected the adeninylalkylcobalamins are unable to function as coenzyme in the ribonucleotide reductase system of L. leichmannii. Furthermore incubation of these cobalamins with excess ribonucleotide reductase, dihydrolipoate and dGTP does not generate the highly resolved ESR spectrum, indicative of the homolytic cleavage of the carbon-cobalt bond [2,14]. However, all five adeninylalkylcobalamins are inhibitors of ATP reduction in the presence of adenosylcobalamin as coenzyme a n d also of hydrogen exchange between [5'-3H2]adenosylcobalamin and water. The kinetic data were fit to equations for linear competitive and linear non-competitive inhibition. Better fits were obtained for linear non-competitive inhibition. The data summarized in Table III show that the length of the carbon chain interposed between the adenine moiety and the cobalt atom has a profound effect on the enzyme-cobalamin interaction. For instance the Kis values of adeninylpentylcobalamin are almost 20 times smaller than the corresponding Km values for reduction and exchange (4.1 -+ 0.3 and 5.0 + 0.5 pM, respectively). The kinetic data suggest that a five carbon chain between the adenine moiety and the cobalt atom is optimal for enzyme~cobalamin interaction. Lengthening the chain to six carbons or shortening it to three or four carbon weakens the interaction between ribonucleotide reductase and the coenzyme analog. On the other hand the inhibition constants of adeninylethylcobalamin indicate that its interaction with the enzyme is similar to that of adeninylbutylcobalamin. A comparison of the Corey-Pauling-Koltun models of adenosylcobalamin, adeninylbutylcobalamin, adeninylpentylcobalamin and adeninylhexylcobalamin demonstrate that 4, 5 and 6 carbon chains allow the positioning of the adenine moiety over ring C of the corrin nucleus. The better interaction between the enzyme and adeninylpentylcobalamin than that between the enzyme and adenosylcobalamin suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the enzyme upon binding. In contrast the high kinetic constants for adeninylpropylcobalamin suggest that the three carbon chain does not allow proper interaction of both the adenine moiety and the corrin ring with the enzyme. Surprisingly adeninyl-
T A B L E III INHIBITION
PROPERTIES
Inhibition constants
OF ADENINYLALKYLCOBALAMINS
w i t h s t a n d a r d errors.
N u m b e r of methylene groups
Kis (pM)
Reduction
Exchange
Reduction
Exchange
2 3 4 5 6
1 6 . 1 -+ 2 . 4 7 6 . 9 +- 9 . 5 1 6 . 1 +- 2 . 8 0.24-+ 0.04 4.5 +-0.8
1 1 . 8 -+ 1 . 5 7 8 . 7 -+ 1 2 . 6 1 1 . 2 -+ 1 . 6 0.17-+ 0.02 6 . 3 -+ 2 . 5
62.9-+ 7.5 333 +- 4 0 105 -+ 2 2 4.0-+ 1.5 2 1 . 9 +- 4 . 5
5 3 . 8 - + 5.9 246 -+ 3 0 134 + 29 5.8 + 2.8 10.6-+ 2.5
K i i QuM)
232 ethylcobalamin interacts with the enzyme as well as the analog containing a four carbon arm, suggesting that the enzyme is able to accommodate rather drastic changes in its coenzyme. A Corey-Pauling-Koltun model of adeninylethylcobalamin demonstrates that the least hindered position for the adenine moiety is in a plane perpendicular to the corrin ring, while in adenosylcobalamin the adenine moiety lies in a plane at an angle o f 20.5 ° with the plane of the four corrin ring nitrogens. A study involving the interaction between these adeninylalkylcobalamins and other adenosylcobalamin-dependent enzymes, such as dioldehydrase and methylmalonyl-CoA mutase, is presently in progress.
Acknowledgment This work was supported by U.S. Public Health Research Grant GM-20307 from the National Institute of Health.
References 1 Hogenkamp, H.P.C. and Sando, G.N. (1974) Struct. Bonding 20, 23--58 2 Sando, G.N., Blakley, R.L., Hogenkamp, H.P.C. and Hoffmann, P.J. (1975) J. Biol. Chem., in the press 3 Gleason, F.K. and Hogenkamp, H.P.C. (1971) Methods EnzymoL 18, 65--71 4 Hogenkamp, H.P.C. Pailes, W.H. and Brownson, C. (1971) Methods Enzymol. 18, 57--65 5 Carraway, K.L., Huang, P.C. and Scott, T.G. (1968) Synthetic Procedures in Nucleic Acid Chemistry (Zorbach, W.W. and Tipson, R.S., eds.), Vol. 1, pp. 3--5, Interscience, New York 6 Hogenkamp, H.P.C. (1974) Biochemistry 13, 2 7 3 6 - - 2 7 4 0 7 Ladd, J.N., Hogenkamp, H.P.C. and Barker, H.A. (1961) J. Biol. Chem. 236, 2114--2118 8 Friedrich, W. (1964) Biochemisches Taschenbuch (Raven, H.M., ed.), Vol. 1, pp. 708--714, Springer Verlag, Berlin 9 Bray, G.A. (1960) Anal. Bioehem. 1, 279--285 10 Blakley, R.L. ( 1 9 6 6 ) J . Biol. Chem. 241, 176--179 11 Orr, M.D., Blakley, R.L. and Panagou, D. (1972) Anal. Biochem. 45, 68--85 12 Hogenkamp, H.P.C., Ghambeer, R.K., Brownson, C., Blakley, R.L. and Vitols, E. (1968) J. Biol. Chem. 243, 799--808 13 Hogenkamp, H.P.C., Rush, J.E. and Swenson, C.A. (1965) J. Biol. Chem. 240, 3 6 4 1 - - 3 6 4 4 14 Hamilton, J.A., Yamada, R., Blakley, R.L., Hogenkamp, H.P.C., Looney, F.D. and Winfield, M.E. (1971) Biochemistry 10, 347--355
Biochimica et Biophysica Acta, 428 (1976) 228--232
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27829
THE INTERACTION OF ADENINYLALKYLCOBALAMINS WITH RIBONUCLEOTIDE REDUCTASE
GLORIA N. SANDO, MICHAEL E. GRANT and HARRY P.C. HOGENKAMP Department of Biochemistry, College of Medicine, University of Iowa, Iowa City, Iowa 52242 (U.S.A.)
(Received August 26th, 1975)
Summary Several structural analogs of adenosylcobalamin, containing 2, 3, 4, 5 and 6 methylene carbons instead of the ribofuranose moiety, have been synthesized and their interaction with ribonucleotide reductase from Lactobacillus leichmannii has been investigated. Kinetic studies of the inhibition of the reductase by these analogs showed that the adeninylalkylcobalamins with 4, 5 and 6 carbons interposed between the adenine moiety and the cobalt atom are potent inhibitors of ribonucleotide reduction. The stronger interaction between adeninylpentylcobalamin and the enzyme than that between adenosylcobalamin and the enzyme suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the protein upon binding.
Introduction Ribonucleotide reductase catalyzes the irreversible reduction of ribonucleotides to 2'-deoxyribonucleotides. The reductases from several microorganisms, including that of Lactobacillus leichmannii, have an absolute requirement for adenosylcobalamin as a coenzyme [1]. Recently we have examined a series of analogs of adenosylcobalamin f o r their effects on reactions catalyzed by ribonucleotide reductase of L. leichmannii [2]. The results of that study indicated that relatively small changes in either the nucleoside base or the sugar moiety attached to cobalt in the sixth coordination position profoundly affect coenzymic activity. One of these analogs, adenosylethylcobalamin, with two extra methylene groups interposed between the 5'-deoxyadenosine moiety and the cobalt atom was a very potent inhibitor of ribonucleotide reduction. Analogs in which the ribose moiety was replaced by three or four methylene carbons were inhibitory to a lesser extent, suggesting an effect of the carbon chain-length on the ability to interact with the enzyme. These results led us to synthesize a series of analogs of adenosylcobalamin in which the ribofuranose
229 moiety is replaced by 2, 3, 4, 5 and 6 methylene carbons. This communication describes these syntheses as well as the action of the analogs as inhibitors of ATP reduction by the ribonucleotide reductase of L. leichmannii. Materials and Methods
Materials. Cyanocobalamin and 1-bromo-5-chloropentane were obtained from Sigma Chemical Co.; 1-bromo-2-chloroethane, 1-bromo-3-chloropropane, 1-bromo-4-chlorobutane and 1-bromo-6-chlorohexane were supplied by Aldrich and J.T. Baker Chemical Co. Ribonucleotide reductase from L. leichmannii was kindly supplied by Dr. R.L. Blakley of the University of Iowa. [5'-3H~ ] Adenosylcobalamin (22 Ci/mol) and adenosylcobalamin were prepared as described before [ 3,4]. The 9-(w-chloroalkyl)adenines and adeninylalkylcobalamins were prepared by published procedures [5,6]. Methods. Ultraviolet and visible spectra were recorded on a Cary Model 15 spectrophotometer. Other absorbance measurements were made with a Zeiss PM Q II spectrophotometer. Ionization constants were determined by the spectral method of Ladd et al. [7]. Molar extinction coefficients of the cobalamins are based on e36 s = 30.8" 103 M -1 • cm -I for dicyanocobalamin [8]. The cobalamins were converted to dicyanocobalamin by photolysis in the presence of 0.1 M KCN. Descending chromatography on Whatman No. 1 paper was conducted with the following solvent systems: solvent I, sec-butylalcohol/ water/NH4OH (50 : 36 : 14, v/v); II, 1-butanol/ethanol/water (50 : 15 : 35, v/v); III, 1-butanol/isopropyl alcohol/water (37 : 26 : 37, v/v). Radioactivity measurements were made with a Packard Model 2420 TriCarb liquid scintillation spectrometer [9]. Assay procedures. The ability of the adeninylalkylcobalamins to function as coenzymes or inhibitors in the ribonucleotide reductase reaction was determined by two methods. Ribonucleotide reductase activity was determined by measuring the a m o u n t of dATP formed from ATP by the diphenylamine procedure of Blakley and co-workers [10,11]. Reaction mixtures contained 0.5 M potassium phosphate (pH 7.5), 30 mM dithiothreitol, 10 mM ATP, adenosylcobalamin and/or adeninylalkylcobalamin and ribonucleotide reductase (25 pg with a specific activity of approx. 150 units/mg) in a 0.5 ml volume. The reaction was initiated by the addition of all other components to a solution of the cobalamin. After a 10-min incubation of the reaction mixtures in the dark at 37°C, the tubes were placed in ice and the a m o u n t of dATP formed determined [2]. Ribonucleotide reductase activity was also determined by measuring tritium exchange between [ 5'- 3H2 ] adenosylcobalamin and water [ 12]. The reaction mixtures were identical to those described above, except that labeled adenosylcobalamin and less ribonucleotide reductase (0.4--0.8 pg) was used. Steady-state kinetic constants were determined as described before [2]. Results and Discussion
Physical and chemical properties of the adeninylalkyl cobalamins The adeninylalkylcobalamins containing 2, 3, 4 and 5 methylene groups are readily crystallized from 90% aqueous acetone, however, adeninylhexylcobala-
23O TABLE I PAPER CHROMATOGRAPHIC
PROPERTIES OF ADENINYLALKYLCOBALAMINS
N u m b e r of m e t h y l e n e
R ( c y a n o c o b a l a m i n values in i n d i c a t e d s o l v e n t s )
groups
Solvent I
S o l v e n t II
S o l v e n t III
2 3 4 5 6
1.10 0.63 0.76 1.28 1.90
1.03 0.78 0.98 1.15 1.26
1.07 0.93 0.97 1.18 1.28
min is quite soluble in acetone and was crystallized from a concentrated aqueous solution. All crystalline preparations were chromatographically homogeneous in solvents I, II and III (Table I). The absorption spectra of the adeninylalkylcobalamins are presented in Table II. The spectra of the cobalamins with two and three methylene groups interposed between the adenine moiety and the cobalt atom are very similar to that of adenosylcobalamin with a bands at 524 and 522 nm, respectively. In contrast the spectra above 400 nm of the cobalamins containing 4, 5 and 6 methylene groups resemble that of ethylcobalamin with broad maxima at 507, 515, and 509 nm. As in the case of adenosylcobalamin, acidification of aqueous solutions of the adeninylalkytcobalamins causes a spectral shift to lower wavelength. The pKa values estimated from the midpoints of these spectral changes reflect the positive inductive effect of the methylene groups (Table II). Thus the pK a of adeninylethylcobalamin {2.7) is very similar to that of methylcobalamin, while the PKa of the highest homologue, adeninylhexylcobalamin, is similar to that of ethylcobalamin (3.9) [13]. The stability of the carbon-cobalt bond of the adeninylalkylcobalamins is like that of the simple alkylcobalamins. Aqueous solutions of the adeninylalkylcobalamins are readily photolyzed especially in the pres-
T A B L E II ABSORPTION SPECTRA OF ADENOSYLCOBALAMIN
AND ADENINYL ALKYLCOBALAMINS
P o s i t i o n o f m a i n a b s o r p t i o n b a n d is given in n m . M o l a r e x t i n c t i o n c o e f f i c i e n t s (X 1 0 - 3 ) are given in parentheses.
N a t u r e of the sixth l i g a n d AdenosylAdeninyl Adeninyl Adeninyl Adeninyl Adeninyl
0.1 M HC1
266 (41.1) ethyl265 (43.7) propyl- 266 (40.8) butyl- 264 (38.8) pentyl- 265 (41.4) hexyl- 265 (37.3)
287 (23.9) 285 (24.6) 287 (23.5) 287 (22.1) 286 (23.0) 286 (21.3)
306 380 458 (22.2) (8.8)(9.4) 305 315 460 (19.9) (19.9) (9.9) 306 380 460 (23.8) (9.2) (9.6) 304 380 455 (25.7) (9.7) (9.0) 305 382 460 (27.3) (8.8) (8.4) 305 381 464 (24.9) (7.7) (7.9)
0.1 M p h o s p h a t e b u f f e r ( p H 7.0)
pK a
262 (35.1) 263 (38.1) 266 (34.4) 266 (31.8) 265 (33.0) 265 (33.4)
3.5
290 (18.2) 290 (18.7) 290 (18.6) 292 (17.5) 291 (17.6) 290 (18.9)
318 (13.0) 320 (12.4) 319 (13.9) 319 (14.4) 317 (14.5) 305 (14.8)
341 376 522 (12.8) (11.0) (8.0) 340 375 524 (13.4) (11.4) (8.9) 340 377 522 (13.2) (11.1) (8.2) 346 380 507 (12.7) (9.6) (8.3) 345 380 515 ( 1 2 . 5 ) (9.1) ( 9 . 4 ) 345 380 509 (11.6) (7.9) (7.9)
2.7 3.2 3.6 3.7 3.9
231
ence of oxygen, but they are not decomposed by acid (0.1 M HC1 at 90°C for 1 h) or by 0.1 M KCN.
Coenzyme activity of the adeninylalkylcobalamins As expected the adeninylalkylcobalamins are unable to function as coenzyme in the ribonucleotide reductase system of L. leichmannii. Furthermore incubation of these cobalamins with excess ribonucleotide reductase, dihydrolipoate and dGTP does not generate the highly resolved ESR spectrum, indicative of the homolytic cleavage of the carbon-cobalt bond [2,14]. However, all five adeninylalkylcobalamins are inhibitors of ATP reduction in the presence of adenosylcobalamin as coenzyme a n d also of hydrogen exchange between [5'-3H2]adenosylcobalamin and water. The kinetic data were fit to equations for linear competitive and linear non-competitive inhibition. Better fits were obtained for linear non-competitive inhibition. The data summarized in Table III show that the length of the carbon chain interposed between the adenine moiety and the cobalt atom has a profound effect on the enzyme-cobalamin interaction. For instance the Kis values of adeninylpentylcobalamin are almost 20 times smaller than the corresponding Km values for reduction and exchange (4.1 -+ 0.3 and 5.0 + 0.5 pM, respectively). The kinetic data suggest that a five carbon chain between the adenine moiety and the cobalt atom is optimal for enzyme~cobalamin interaction. Lengthening the chain to six carbons or shortening it to three or four carbon weakens the interaction between ribonucleotide reductase and the coenzyme analog. On the other hand the inhibition constants of adeninylethylcobalamin indicate that its interaction with the enzyme is similar to that of adeninylbutylcobalamin. A comparison of the Corey-Pauling-Koltun models of adenosylcobalamin, adeninylbutylcobalamin, adeninylpentylcobalamin and adeninylhexylcobalamin demonstrate that 4, 5 and 6 carbon chains allow the positioning of the adenine moiety over ring C of the corrin nucleus. The better interaction between the enzyme and adeninylpentylcobalamin than that between the enzyme and adenosylcobalamin suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the enzyme upon binding. In contrast the high kinetic constants for adeninylpropylcobalamin suggest that the three carbon chain does not allow proper interaction of both the adenine moiety and the corrin ring with the enzyme. Surprisingly adeninyl-
T A B L E III INHIBITION
PROPERTIES
Inhibition constants
OF ADENINYLALKYLCOBALAMINS
w i t h s t a n d a r d errors.
N u m b e r of methylene groups
Kis (pM)
Reduction
Exchange
Reduction
Exchange
2 3 4 5 6
1 6 . 1 -+ 2 . 4 7 6 . 9 +- 9 . 5 1 6 . 1 +- 2 . 8 0.24-+ 0.04 4.5 +-0.8
1 1 . 8 -+ 1 . 5 7 8 . 7 -+ 1 2 . 6 1 1 . 2 -+ 1 . 6 0.17-+ 0.02 6 . 3 -+ 2 . 5
62.9-+ 7.5 333 +- 4 0 105 -+ 2 2 4.0-+ 1.5 2 1 . 9 +- 4 . 5
5 3 . 8 - + 5.9 246 -+ 3 0 134 + 29 5.8 + 2.8 10.6-+ 2.5
K i i QuM)
232 ethylcobalamin interacts with the enzyme as well as the analog containing a four carbon arm, suggesting that the enzyme is able to accommodate rather drastic changes in its coenzyme. A Corey-Pauling-Koltun model of adeninylethylcobalamin demonstrates that the least hindered position for the adenine moiety is in a plane perpendicular to the corrin ring, while in adenosylcobalamin the adenine moiety lies in a plane at an angle o f 20.5 ° with the plane of the four corrin ring nitrogens. A study involving the interaction between these adeninylalkylcobalamins and other adenosylcobalamin-dependent enzymes, such as dioldehydrase and methylmalonyl-CoA mutase, is presently in progress.
Acknowledgment This work was supported by U.S. Public Health Research Grant GM-20307 from the National Institute of Health.
References 1 Hogenkamp, H.P.C. and Sando, G.N. (1974) Struct. Bonding 20, 23--58 2 Sando, G.N., Blakley, R.L., Hogenkamp, H.P.C. and Hoffmann, P.J. (1975) J. Biol. Chem., in the press 3 Gleason, F.K. and Hogenkamp, H.P.C. (1971) Methods EnzymoL 18, 65--71 4 Hogenkamp, H.P.C. Pailes, W.H. and Brownson, C. (1971) Methods Enzymol. 18, 57--65 5 Carraway, K.L., Huang, P.C. and Scott, T.G. (1968) Synthetic Procedures in Nucleic Acid Chemistry (Zorbach, W.W. and Tipson, R.S., eds.), Vol. 1, pp. 3--5, Interscience, New York 6 Hogenkamp, H.P.C. (1974) Biochemistry 13, 2 7 3 6 - - 2 7 4 0 7 Ladd, J.N., Hogenkamp, H.P.C. and Barker, H.A. (1961) J. Biol. Chem. 236, 2114--2118 8 Friedrich, W. (1964) Biochemisches Taschenbuch (Raven, H.M., ed.), Vol. 1, pp. 708--714, Springer Verlag, Berlin 9 Bray, G.A. (1960) Anal. Bioehem. 1, 279--285 10 Blakley, R.L. ( 1 9 6 6 ) J . Biol. Chem. 241, 176--179 11 Orr, M.D., Blakley, R.L. and Panagou, D. (1972) Anal. Biochem. 45, 68--85 12 Hogenkamp, H.P.C., Ghambeer, R.K., Brownson, C., Blakley, R.L. and Vitols, E. (1968) J. Biol. Chem. 243, 799--808 13 Hogenkamp, H.P.C., Rush, J.E. and Swenson, C.A. (1965) J. Biol. Chem. 240, 3 6 4 1 - - 3 6 4 4 14 Hamilton, J.A., Yamada, R., Blakley, R.L., Hogenkamp, H.P.C., Looney, F.D. and Winfield, M.E. (1971) Biochemistry 10, 347--355
