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The carboxylation of biotin
ARCHIVES
OF BIOCHEMISTRY
AND
183,
BIOPHYSICS
189-199
The Carboxylation Substrate
Recognition
and Activation WILLIAM
Institute
for Cancer
Research,
The Fox
of Biotin
by Complementary
Hydrogen
Bonding
C. STALLINGS
Chase
Received
(1977)
Cancer January
Center,
Philadelphia,
Pennsylvania
19111
19, 1977
Analysis of the hydrogen bonding observed in the crystal structures of biotin, several of its derivatives, and some urea complexes has resulted in the proposal of a mechanism for biotin recognition of bicarbonate. The recognition is by a system of complementary hydrogen bonding between bicarbonate and the ureido-carbonyl oxygen and the nitrogen tram to the valeric acid side chain of biotin: this is illustrated below.
This mechanism of recognition suggests a mechanism for the nucleophilic activation of biotin which incorporates keto-enol tautomerism of the ureido moiety. This is consistent with data regarding the chemical reactivity of some model compounds. The mechanisms of recognition and activation provide an example of substrate activation and may have a more general applicability to other enzymatic reactions.
D-(+)-Biotin is a naturally occurring coenzyme which participates in enzymatic carboxylation, transcarboxylation, and decarboxylation reactions. The molecule acts by binding carbon dioxide and then transferring it to other molecules. As an enzymatic cofactor, biotin is involved in vital processes which include fatty acid synthesis, gluconeogenesis, and amino acid metabolism. The molecular structure of this coenzyme is illustrated below. The symmetry of the bicyclic ring system is broken by the one long side chain (the valeric acid side chain). Copyright All rights
C 1977 by Academic Press, Inc. of reproduction in any form reserved.
100 0
,i; ,” 2’ HN 3’ \
1’ NH ’
In its biochemical action, a biotin molecule is covalently bound to an enzyme through an amide linkage between the valeric acid side chain of biotin and the Eamino group of an enzymatic lysine resi-
ISSN
0003-9861
190
WILLIAM
C. STALLINGS
due. Enzymes for which biotin has been shown to be a prosthetic group include acyl-coenzyme A carboxylases (acetyl-, propionyl-, /?-methylcrotonyl-, geranyl-), pyruvate carboxylase, ATP-urea amidolyase , methylmalonyl-coenzyme A: pyruvate transcarboxylase, methylmalonyl-coenzyme A decarboxylase, and oxaloacetate decarboxylase. These systems have been extensively reviewed by Moss and Lane (1). Biotin participation in enzymatic carboxylation is represented by a two-step reaction sequence. Enzyme-biotin + HCOBp + ATP MB G enzyme-biotin-CO,+ ADP Enzyme-biotin-CO,-
114
+ Pi
+ acceptor
it carboxylated
acceptor
I lb1
+ enzyme-biotin In the case of carboxylation, Reaction [la] describes the carboxylation of biotin by some species of CO, which is generally believed to be bicarbonate; this reaction also involves ATP and a divalent metal ion. In the subsequent reaction, Ilbl, CO, is transferred to an acceptor which may be an acyl-CoA, pyruvate, or urea. In the case of transcarboxylation, bicarbonate (in Reaction [la]) is replaced by methylmalonyl-CoA which carboxylates biotin. CO, is then transferred finally to pyruvate, the acceptor of Reaction [lb]. In the case of decarboxylation, the reactions may be thought of in terms of enzymes which catalyze the reverse of Reactions [la] and llbl. It appears that biotin-dependent enzymes are high molecular weight (-6 x 10” and greater) multisubunit structures and recent studies (2) of Escherichia coli acetyl-CoA carboxylase have indicated that Reactions [la] and Ilbl occur at distinct sites on different subunits of the enzyme. Three purified subunits of this enzyme were studied: biotin carrier protein which contains the lysine residue by which biotin is anchored to enzyme, biotin carboxylase which contains the active site of Reaction [la], and carboxyl transferase
which contains the active site of Reaction [lb]. Since the extension of the Valery1 side chain linked to the lysine residue has been estimated to be 14 A, a “swinging arm mechanism” evolved to explain biotin translocation between the two active sites. More recently, however, it has been demonstrated (3) that for transcarboxyiases isolated from Propionibacterium shermanii, the active sites, although located on different subunits, are separated by no more than 7 A. As a result, it has been postulated that the 14-A arm is primarily necessary to extend into the area between the interface of the three subunits and that translocation plays a role in the overall reaction mechanism, which is at best minimal. Although mechanisms for both the carboxylation and the decarboxylation of biotin have been proposed, a detailed mechanism (i.e., one which incorporates detailed aspects of the molecular geometry involved) for the interaction between biotin and bicarbonate has remained lacking. I attempt to provide some details in a mechanism proposed later in this article. While the question regarding which species of YJOr” (i.e., COZ, CO:,” , HCO:, , or H&O,,) remains strictly unresolved, most of the evidence currently available favors attack by bicarbonate. Such studies include experiments involving IHO-labeled bicarbonate (4) and equilibrium studies (5) carried out at temperatures below 15°C using only H,O and CO, as initial reactants. Since it is known that, at these temperatures, the CO, it H&O2 s HCO:, equilibrium is not obtained in less than 1 min, the generation of bicarbonate may be made rate limiting for the carboxylation of biotin. It was first demonstrated by Kaziro et al. (4) using pig heart propionyl-CoA carboxylase and lXO-labeled bicarbonate that, for each lx0 found incorporated in P,. two were found incorporated in the final product, methylmalonyl-CoA. These isotopelabeling studies, which also demonstrated HCO:, - and ADP-dependent ATP-““P, exchange and HCO:,-and Pi-dependent ATP-] ‘CJADP exchange, resulted in the proposal of a reaction mechanism for the carboxylation of biotin. This mechanism is
THE
CARBOXYLATION
mechaillustrated in Fig. la. Another nism, not unlike that in Fig. la but incorporating greater detail, is that of McClure et al. (6), which is illustrated in Fig. lb. This is a concerted mechanism whereby the biotin-HCOx~ reaction is concomitant with an ATP-HCO,,reaction. As pointed out by Kaziro et al., these results are also consistent with a stepwise mechanism involving the formation of an activated bicarbonate, perhaps carbonyl phosphate 0 II (HO-C-OPO,,‘-), which in turn carboxylates biotin. Other reaction mechanisms proposed include a stepwise mechanism whereby the biotin-bicarbonate reaction is preceded by phosphorylation of the enzyme. This mechanism is in accord with the experimental finding (7) that pyruvate carboxylase, purified from chicken liver mitochondria (unlike the acyl-CoA carboxylases), catalyzes a slow ATP- I ’ ‘C!IADP exchange which apparently proceeds without biotin participation; the investigators themselves point out that an abortive pathway is consistent with their results. Because of the ATP-:‘“P, and ATPATP + HCC$
OF
191
BIOTIN
I “CIADP exchanges and their dependencies, the stepwise mechanisms require an ordered release of Pi and ADP which may not precede the carboxylation of biotin. Finally, Bruice and Hegarty (8) have suggested the possibility that the initial attack on bicarbonate is by the ureido-carbony1 oxygen of biotin. In the course of the discussion which follows, the reaction mechanism outlined in Fig. la will be elaborated upon. STRUCTURAL RECOGNITION
INFORMATION ON OF BICARBONATE
Recently, the crystal structure of D-(+ )biotin has been determined (9) by the single crystal X-ray diffraction technique, affording a precise description of the molecular geometry of this coenzyme. A view of the packing and hydrogen bonding is given in Fig. 2a. Of particular interest in the crystal structure is the intermolecular hydrogen bonding between the two oxygen atoms of the carboxylic acid group of the valeric acid side chain and both the carbony1 oxygen and Nl’ of the ureido group of another molecule. The carboxylic acid functionality is not ionized so that one of
+ Enzyme-botn
R~tme ‘0 / P=O
0
-0’
FIG.
la.
Concerted
‘0 / PC0
!? HN’
BIOTIN
HN:
mechanism
of Kaziro
et al.
(4)
adon~n~
192
WILLIAM
C
STALLINGS
RI++
FIG.
lb.
More
detailed
concerted
mechanism
-0
/\
,o P=O
of McClure
et al.
(6).
FIG. 2a. The packing in the crystal structure of n-(+I-biotin is illustrated. Nl’ and 02’ of one molecule hydrogen bond with OlOA and OlOB (the carboxylic oxygens) of a neighboring molecule. (OlOA, hydrogen bound to 02’, is protonated.) OlOB is also the acceptor in an N3’ OlOB intermolecular hydrogen bond.
its oxygen atoms is covalently linked to a hydrogen atom and is thus capable of being a donor in a hydrogen bond with 02’ of the ureido ring. Biotin attaches CO, at
Nl’, attack by N3’ being hindered presumably for steric reasons involving the proximity of the valeric acid side chain. This scheme of hydrogen bonding is probably a
THE
CARBOXYLATION
5‘
OF
193
BIOTIN
16
FIG. 2b. Illustrated
are scale diagrams of structural data relevant to biotin recognition of bicarbonate. These were produced by calculating a least-squares plane through the ureido or urea carbon, nitrogen, and oxygen atoms. Deviations (in Angstrom units) from this plane are indicated when greater than 0.05 A. Hydrogen bond donor-acceptor distances are also included. (1) biotin; (2) oxybiotin; (3) dethiobiotin (in this case, complementary hydrogen bonding occurs between 02’ and N3’ of one molecule and the carboxylic acid functionality of a neighboring molecule); (4) urea-oxalic acid complex; (5) urea-phosphoric acid complex; (6) a relevant portion of the his-p-bromoanilide of CO, biotin which serves as a model of the reaction product of biotin and bicarbonate.
good model for the ureido recognition of bicarbonate since the carboxylic acid functionality in many ways resembles bicarbonate. Similar schemes of hydrogen
bonding are also possible and have been shown to exist in the recently determined crystal structures of dethiobiotin (10) and oxybiotin (11). The crystal structures of
194
WILLIAM
C. STALLINGS
urea complexed with oxalic acid (12) and phosphoric acid (13) also bear upon this point because of the similarity between urea and the ureido portion of biotin. In these structures, a similar 0. . . , NH.. .-. . .HO, . . .O hydrogen bonding pattern also occurs. Scale diagrams showing portions of the hydrogen bonding in each of these structures are presented in Fig. 2b. It would seem evident, then, that the ureido group possesses a geometry which is ideal for complementary hydrogen bonding with a protonated acid. This may be illustrated as
A model for biotin recognition of bicarbonate via hydrogen bonding is therefore proposed and this model is illustrated in Fig. 3. Relevant points include complementary hydrogen bonding between the ureido-carbony1 oxygen with the protonated oxygen of bicarbonate and Nl’ of the ureido group with an unprotonated oxygen of bicarbonate. This scheme of complementary hydrogen bonding is somewhat analogous to the complementarity in hydrogen bonding patterns observed in nucleic acids. A PROPOSED
MECHANISM ACTIVATION
OF
BIOTIN
This mode of biotin recognition of bicarbonate suggests a likely mechanism for the carboxylation reaction, [la]. The hydrogen bonded complex illustrated in Fig. 3 is probably stable, and it is the additional approach of ATP, providing chemical potential for the reaction, which triggers carboxylation at Nl’ by inducing the electronic rearrangements illustrated in Fig. 4; the mechanism presented here compares closely with that of Fig. la. In this mechanism, polarization of the car-bony1 bond, with corresponding enhanced nucleophilicity at Nl’ (the tautomerism of the ureido group, illustrated in Fig. 4b), results from hydrogen bonding with bicarbonate. This is a crucial step of the mechanism since, as has been pointed out (14), the nucleophilicity of Nl’ is prob-
FIG. 3. Proposed biotin-bicarbonate via hydrogen bonding.
recognition
ably very low, thus necessitating some form of biotin activation to enhance the nucleophilicity at this nitrogen. Hegarty et al. (151, having studied the reactivity of 2methoxy-2-imidazoline as a model for the enol form of biotin, found the nucleophilicity of this compound to be greatly enhanced relative to that of 2-imidazolidone, a model for the keto form of biotin. They therefore suggested that a probable form of activated biotin is the enol tautomer. It is perhaps interesting to note that, in the mechanism proposed here, tautomeric activation results from interaction with bicarbonate, the species with which biotin finally reacts. It is suggested that the activation consists of two proton transfers with resultant tautomerism of the ureido group. One proton is transferred from the protonated oxygen of bicarbonate to the ureido oxygen of biotin. Another proton is transfered from Nl’ of biotin to an oxygen of bicarbonate. Hence, the protons are transferred from their hydrogen bond donors to their respective hydrogen bond acceptors; the hydrogen bond donors of Fig. 4a become the hydrogen bond acceptors of Fig. 4b. The keto and enol tautomers, illustrated, respectively, in Figs. 4a and 4b, are probably in rapid equilibrium. The approach of ATP, indicated in Fig. 4b, shifts the equilibrium to the enol tautomer by reacting with the bicarbonate oxygen which is hydrogen-bonded to Nl’ of biotin; as indicated in Figs. 4b and 4c, this triggers the completion of the reaction. In the crystal structure of the urea-phosphoric acid com-
FIG. 4. Proposed reaction mechanism. Although a concerted reaction mechanism is suggested, it is illustrated in three parts for clarity. (ai Recognition of biotin by bicarbonate. Hydrogen bonding of bicarbonate to carbonyl oxygen and Nl’ of biotin as shown by the data discussed in this paper. tb) Tautomerism as a result of hydrogen bonding. Polarization of the carbonyl group and increased nucleophilicity at Nl’. ATP participation at this point shifts the equilibrium to the enol tautomer by reacting with the bicarbonate oxygen which is involved in the hydrogen bond with Nl’ of biotin. The ATP conformation illustrated has been observed crystallographically (26). (c) Covalent bond formation between Nl’ of biotin and C of bicarbonate. Formation of P, and ADP from ATP.
196
WILLIAM
C. STALLINGS TABLE
GEOMETRIC
~.~-.~ Uranium
DATA Substance
RELEVANT
TO
THE POLARIZATION Reference
~ nitrate
I AND TAUTOMERISM Has the oxygen been protonated?
OF c-o
THE
(A)
16
Yes
1.298 (1)
17
Yes
1.301 (4)
13
Partially
1.281 (3,
12
No
1.261 (3)
Urea
21
NO
1.270 (7)
N-Methylurea
22
No
1.248 (1)
9
No
1.249
(6)
23
No
1.236
(6)
Dethiobiotin
10
No
1.244
(4)
Oxybiotin
11
No
1.270
23
No
1.243
N-Methyluronium Urea-phosphoric Urea-oxalic
nitrate acid
complex
acid complex
Biotin Biotin
Azabiotin
methyl
ester
hydrochloride
hydrate
UREIDO
GROUP" C-N
(11
1.312 1.315 1.309 1.307 1.324 1.323 1.329 1.329 1.326 1.326 1.340 1.336 1.332 1.351 1.350 1.351 1.346 1.346 1.328 1.290 1.345 1.348 1.37
(1) (1) (41 (4) (2) (2) (4) (6, (6) (6) (2, (2) (6, (71 (7) (6) (4) (41
12 barbiturate structures averaged 24 No 1.21 -~~-~~ ” This table indicates that, in general, a tautomeric-like geometry is observed upon ureido-oxygen protonation. It further illustrates that in biotin, as in urea, the polarized (tautomeric) forms of their respective resomers contribute significantly to the overall electronic structures. The polarized forms, however, do not always contribute as significantly in a ureido system since a recent survey (24) of 12 barbiturate structures (barbiturates contain a ureido-like chemical unit) indicates the presence of unpolarized carbonyl C=O bonds. It is concluded, then, that biotin and urea are similar in their polarizability but that not all molecules containing the ureido group have the ability to be similarly polarized. This has been discussed more fully elsewhere (9, IO). For comparison, reference C-O, C=O, C-N, and C=N bond lengths are quoted (25): C=O, 1.21 A; C-O, 1.43 A; C=N, 1.25 A; C-N, 1.47 A.
plex’ (13), the hydrogen atom involved in the hydrogen bond between the urea carbony1 functionality and a protonated oxygen of H,PO, is actually equidistant (1.223 and 1.207 A) from these two oxygen atoms, indicative of the possibility (see Fig. 4b) of protonation of the ureido oxygen in biotin as a result of the proposed hydrogen bonding scheme with bicarbonate. In the crystal structure of uronium nitrate (see Footnote 1) (16) and N-methyluronium nitrate (171, the proton has actually been transferred to the carbonyl oxygen although ’ These structures have been determined by the technique of neutron diffraction such that the hydrogen atoms have been located with precision not obtainable using the X-ray technique.
complementary hydrogen bonding (as described) is not observed. Changes which occur, upon protonation, in the C-O and C-N bond lengths are significant, indicating extensive polarization of the C-O and double-bond character in the C-N bond, the electronic rearrangement suggested in Fig. 4b and triggered by the proposed proton transfer from bicarbonate to ureido oxygen. Geometric data relevant to the polarization and the tautomerism of the ureido group are summarized in Table I. The result of increased nucleophilicity at Nl’ is covalent bond formation between this nitrogen and the carbon atom of bicarbonate. Concomitantly, the protonated ox-
THE
CARBOXYLATION
ygen of bicarbonate is added to ATP which is cleaved to ADP and Pi. This is illustrated in Fig. 4~. The crystal structure of the bis-p-bromoanilide of carbon dioxide biotin (18) perhaps provides a good model for the final product. The molecular formula of this compound is illustrated below and a scale diagram of its structure has been included in Fig. 2b. O-----H \
II HN
&
P\
i
43
!3r
“-No
s
“*a a
It is interesting to note that the crystal structure contains an intramolecular N. ’ .O hydrogen bond (2.64 A1 between the ureido oxygen and the protonated nitrogen atom of the group attached to Nl’. An intramolecular 0. .O hydrogen bond has therefore been incorporated into Fig. 4c. Although this mechanism does not account for the role of the divalent metal ion, it is not difficult to speculate about its possible function. This might be one of coordination involving the sulfur atom or, perhaps even more likely, it may serve to properly coordinate the triphosphate portion of ATP and bicarbonate in a geometry which induces the carboxylation reaction. This latter idea has been incorporated into the rather detailed mechanism of McClure et al. (6). The mechanism also does not provide a description of the interaction and reaction between bicarbonate and ATP. (It is assumed that ATP is not protonated in this reaction. Otherwise, the ureido group might also be a recognition site for this portion of ATP, especially in view of the existence of a tightly bound urea-triprotonated phosphoric acid complex.) Structural models derived from experimental data are lacking for these cases.
OF
BIOTIN
197
By superimposing the bicyclic ring systems of biotin and the bis-p-bromoanilide of CO,-biotin as illustrated in Fig. 2b, a sense of the dynamics of the reaction mechanism is achieved. This is illustrated in Fig. 5. Bicarbonate must undergo a relative rotation and translation toward Nl’ to accomplish covalent bond formation. The proposed mechanism is in accord with the kinetic isotope exchange data of Kaziro et al. (4), mentioned earlier. Figure 4 illustrates a concerted reaction mechanism involving concomitant biotin-HCO,, and HCO,,--ATP reactions; these reactions are preceded by biotin-HCO,; complexation. A stepwise mechanism involving prior formation of an activated bicarbon-
ate such as HO-C-OPO,,” cannot be excluded on the basis of the arguments presented here since this form of bicarbonate would be capable of similar complementary hydrogen bonding with biotin. Recent studies (19) of sheep kidney pyruvate carboxylase have provided evidence against a stepwise mechanism involving phosphorylation of the enzyme prior to the carboxylation reaction. This mechanism is also in
FIG. 5. Dynamic representation of the reaction mechanism obtained by superimposing the bicyclic ring system of biotin and the bis-p-bromoanilide of CO, biotin as illustrated in Fig. 2b. This diagram illustrates the reactants (biotin and bicarbonate), a model of their reaction product, and the relative dynamics of the proposed reaction mechanism. It is evident that to accomplish covalent bond formation between Nl’ of biotin and C of bicarbonate, hydrogen bound bicarbonate must undergo a relative rotation, indicated by the arrow, and translation toward Nl’. The initial position of bicarbonate is indicated by the letters a, b, c, and d. After covalent bond formation, a, b, and c have moved to a’, b’, and c’ with cleavage of b-d bond; d is transferred to ATP (as d’).
198
WILLIAM
C. STALLINGS
accord with some finer details observed by various investigators. Caplow (14), using 2-imidazolidone as a model for biotin, was unable to demonstrate any reactivity between this compound and such carboxylating agents as p-nitrophenyl acetate, acetylimidazole, and acetyl-3-methylimidazolium chloride. He concluded that, in biotin, the nucleophilicity at Nl’ is probably very low and that, in addition to the possibility of bicarbonate activation, the nucleophilicity at Nl’ must be increased by some form of biotin activation. Note also that the enol form of biotin, which has been suggested as the probable intermediate (15), is involved in the mechanism proposed here. A probable mechanism of activation, suggested by Gressier et al. (20), is via polarization of the ureido-carbonyl group by hydrogen bonding. Such a scheme would stabilize the polarized forms of the biotin resomers and increase the
presumably reacts. The tautomerism of biotin which results from this hydrogen bonding increases the nucleophilicity of Nl’, and the participation of ATP provides chemical potential, driving the reaction to completion. In this way, a covalent bond is formed between Nl’ and the carbon atom of bicarbonate. At the same time, oxygen is added to ATP resulting in its cleavage to ADP and P,. This mechanism represents an example of substrate activation; the binding of bicarbonate, the substrate in question, increases the nucleophilicity of biotin. It is possible that this mechanism is essentially more general. For example, the reaction between citrulline and aspartic acid, catalyzed by argininosuccinate synthetase, may proceed by a similar mechanistic pathway and with a similar pattern of recognition. The reaction also involves ATP. This question is currently being investigated. ACKNOWLEDGMENTS
acidity of the Nl’ proton, leading to enhanced nucleophilicity at Nl’. The recent single crystal studies of biotin (9, 10) have revealed that the carbonyl oxygen is capable of forming a strong hydrogen bond, and an analysis of the bond lengths indicates that the polarized forms of the biotin resomers contribute significantly to the overall electronic structure. SUMMARY
In summary, then, a probable mode of recognition between the ureido portion of biotin and bicarbonate is via a scheme of complementary hydrogen bonding involving the carbonyl group and Nl’ of biotin. This mode of recognition has suggested a mechanism which explains nucleophilic activation at Nl’ of biotin. This activation is via polarization of the ureido-carbonyl group as a result of hydrogen bonding with bicarbonate, the species with which biotin
I would like to thank Dr. Mary Wimmer for helpful conversations. I would like to acknowledge enlightening conversations with Dr. George DeTitta and to thank both him and Dr. R. Parthasarathy for giving me the oxybiotin coordinates prior to publication. I would also especially like to thank Dr. Jenny Glusker for making many valuable suggestions regarding both the form and the content of this paper as well as for the preparation of its figures. This research was supported by USPHS Grants F32 CA-05322, CA-10925, CA-06927, and RR-05539 from the National Institutes of Health, by AG-370 from the National Science Foundation, and by an appropriation from the Commonwealth of Pennsylvania. REFERENCES 1. Moss, J., AND zymol. Relat. 2. (a) GUCHHAIT,
M. D. 11971) Advan. EnAreas Mol. Bid. 35, 321-442. R. B., POLAKIS, S. E., DIMROTH, P., STOLL, E., Moss, J., AND LANE, M. D. (1974) J. Biol. Chem. 249, 6633-6645; (b) GUCHHAIT, R. B., POLAKIS, S. E., HOLLIS, D., FENSELAU, C., AND LANE, M. D. (197415. Bid. Chem. 249, 6646-6656; (cl POLAKIS, S. E., GUCHHAIT, R. B., ZWEIGEL, E. E., AND LANE, M. D. 11974)J. Biol. Chem. 249, 6657-6667. 3. FUNG, C. H., GUPTA, R. K., AND MILDVAN, A. S. (1976) Biochemistry 15, 85-92. 4. KAZIRO, OCHOA,
LANE,
Y., HASS, S. (1962)
L.
F.,
J. Bid.
BOYER,
Chem.
P.
D.,
237,
AND
1460-
THE
CARBOXYLATION
1468. 5. FITMORE, D. L., AND COOPER, T. G. (1970) d. Theor. Biol. 29, 131-145. 6. MCCLURE, W. R., LARDY, H. A., AND CLELAND, W. W. (1971)J. Biol. Chem. 246, 3584-3590. 7. SCRUTTON, M. C., AND UTTER, M. F. (1965) J. Biol. Chem. 240, 3714-3723. 8. BRUICE, T. C., AND HEGARTY, A. F. (1970) Proc. Nat. Acad. Sci. USA 65, 805-809. 9. DETITTA, G. T., EDMONDS, J. W., STALLING, W., AND DONOHUE, J. (1976) J. Amer. Chem. Sot. 98, 1920-1926. 10. CHEN, C. S., PARTHASARATHY, R., AND DETITTA, G. T. (1976) J. Amer. Chem. Sot. 98, 49834990. 11. DETITTA, G. T., AND PARTHASARATHY, R. (1976) Unpublished results. 12. HARKEMA, S., BATS, J. W., WEYENBERG, A. M., AND FEIL, D. (1972) Acta Crystallogr. B28, 1646-1648; see also: HARKEMA, S., BATS, J. W., WEYENBERG, A. M., AND FEIL, D. (19731 Acta Crystallogr. B29, 143. 13. KOSTANSEK, E. C., AND BUSING, W. R. (1972) Acta Crystallogr. R28, 2454-2459. 14. CAPLOW, M. (1965) J. Amer. Chem. Sot. 87, 5774-5785. 15. HEGARTY, A. F., BRUICE, T. F., AND BENKOVIC. S. J. (1969) Chem. Commun. 1173-1174. 16. WORSHAM, J. E., AND BUSING, W. R. (1969)Acta Crystallogr. B25, 572-578.
OF
BIOTIN
199
17. SELMAN, W. F., AND HARKEMA, S. (1971) Progress Report, Chemical Physics Laboratory, Twente University of Technology. 18. BONNEMERE, C., HAMILTON, J. A., STEINRAUF. L. K., AND KNAPPE, J. (1965) Biochemistry 4. 240-245. 19. ASHMAN, L. K., AND KEECH, D. B. (1975)J. Bzol. Chem. 250. 14-21. 20. GRESSIER, R., SIGEL, H., WRIGHT, L. D., AND MCCORMICK, D. B. (1973) Biochemistry 12, 1917-1922. 21. CARON, A., AND DONOHUE, J. (19691 Acta Crystallogr. B25, 404. 22. HUISZOON, C., AND TIEMESSEN, G. W. H. (1976) Acta Crystallogr. B32, 160441606. 23. DETITTA, G. T., GLICK, M., PARTHASARATHY, R., AND STALLINGS, W. (1976) Presented at the American Crystallographic Association Meeting, Northwestern University, Evanston, Ill., Aug. 9-12, 1976, Abstracts. 73-74. 24. CRAVEN, B. M., CUSATIS, C., GARLAND, G. L., AND VIZZINI, E. A. (1973) J. Mol. Struct. 16, 331-342. 25. PAULING, L. (1960) in The Nature of the Chemical Bond, pp. 228-229, Cornell University Press, Ithaca. 26. KENNARD, O., ISAACS, N. W., MOTHERWELL, W. D. S., COPPOLA, J. C., WAMPLER. D. L., LARSON, A. C.. AND WATSON, D. G. (1971) Proc. Roy. Sot. hndon SeF. A 325, 401-436.
OF BIOCHEMISTRY
AND
183,
BIOPHYSICS
189-199
The Carboxylation Substrate
Recognition
and Activation WILLIAM
Institute
for Cancer
Research,
The Fox
of Biotin
by Complementary
Hydrogen
Bonding
C. STALLINGS
Chase
Received
(1977)
Cancer January
Center,
Philadelphia,
Pennsylvania
19111
19, 1977
Analysis of the hydrogen bonding observed in the crystal structures of biotin, several of its derivatives, and some urea complexes has resulted in the proposal of a mechanism for biotin recognition of bicarbonate. The recognition is by a system of complementary hydrogen bonding between bicarbonate and the ureido-carbonyl oxygen and the nitrogen tram to the valeric acid side chain of biotin: this is illustrated below.
This mechanism of recognition suggests a mechanism for the nucleophilic activation of biotin which incorporates keto-enol tautomerism of the ureido moiety. This is consistent with data regarding the chemical reactivity of some model compounds. The mechanisms of recognition and activation provide an example of substrate activation and may have a more general applicability to other enzymatic reactions.
D-(+)-Biotin is a naturally occurring coenzyme which participates in enzymatic carboxylation, transcarboxylation, and decarboxylation reactions. The molecule acts by binding carbon dioxide and then transferring it to other molecules. As an enzymatic cofactor, biotin is involved in vital processes which include fatty acid synthesis, gluconeogenesis, and amino acid metabolism. The molecular structure of this coenzyme is illustrated below. The symmetry of the bicyclic ring system is broken by the one long side chain (the valeric acid side chain). Copyright All rights
C 1977 by Academic Press, Inc. of reproduction in any form reserved.
100 0
,i; ,” 2’ HN 3’ \
1’ NH ’
In its biochemical action, a biotin molecule is covalently bound to an enzyme through an amide linkage between the valeric acid side chain of biotin and the Eamino group of an enzymatic lysine resi-
ISSN
0003-9861
190
WILLIAM
C. STALLINGS
due. Enzymes for which biotin has been shown to be a prosthetic group include acyl-coenzyme A carboxylases (acetyl-, propionyl-, /?-methylcrotonyl-, geranyl-), pyruvate carboxylase, ATP-urea amidolyase , methylmalonyl-coenzyme A: pyruvate transcarboxylase, methylmalonyl-coenzyme A decarboxylase, and oxaloacetate decarboxylase. These systems have been extensively reviewed by Moss and Lane (1). Biotin participation in enzymatic carboxylation is represented by a two-step reaction sequence. Enzyme-biotin + HCOBp + ATP MB G enzyme-biotin-CO,+ ADP Enzyme-biotin-CO,-
114
+ Pi
+ acceptor
it carboxylated
acceptor
I lb1
+ enzyme-biotin In the case of carboxylation, Reaction [la] describes the carboxylation of biotin by some species of CO, which is generally believed to be bicarbonate; this reaction also involves ATP and a divalent metal ion. In the subsequent reaction, Ilbl, CO, is transferred to an acceptor which may be an acyl-CoA, pyruvate, or urea. In the case of transcarboxylation, bicarbonate (in Reaction [la]) is replaced by methylmalonyl-CoA which carboxylates biotin. CO, is then transferred finally to pyruvate, the acceptor of Reaction [lb]. In the case of decarboxylation, the reactions may be thought of in terms of enzymes which catalyze the reverse of Reactions [la] and llbl. It appears that biotin-dependent enzymes are high molecular weight (-6 x 10” and greater) multisubunit structures and recent studies (2) of Escherichia coli acetyl-CoA carboxylase have indicated that Reactions [la] and Ilbl occur at distinct sites on different subunits of the enzyme. Three purified subunits of this enzyme were studied: biotin carrier protein which contains the lysine residue by which biotin is anchored to enzyme, biotin carboxylase which contains the active site of Reaction [la], and carboxyl transferase
which contains the active site of Reaction [lb]. Since the extension of the Valery1 side chain linked to the lysine residue has been estimated to be 14 A, a “swinging arm mechanism” evolved to explain biotin translocation between the two active sites. More recently, however, it has been demonstrated (3) that for transcarboxyiases isolated from Propionibacterium shermanii, the active sites, although located on different subunits, are separated by no more than 7 A. As a result, it has been postulated that the 14-A arm is primarily necessary to extend into the area between the interface of the three subunits and that translocation plays a role in the overall reaction mechanism, which is at best minimal. Although mechanisms for both the carboxylation and the decarboxylation of biotin have been proposed, a detailed mechanism (i.e., one which incorporates detailed aspects of the molecular geometry involved) for the interaction between biotin and bicarbonate has remained lacking. I attempt to provide some details in a mechanism proposed later in this article. While the question regarding which species of YJOr” (i.e., COZ, CO:,” , HCO:, , or H&O,,) remains strictly unresolved, most of the evidence currently available favors attack by bicarbonate. Such studies include experiments involving IHO-labeled bicarbonate (4) and equilibrium studies (5) carried out at temperatures below 15°C using only H,O and CO, as initial reactants. Since it is known that, at these temperatures, the CO, it H&O2 s HCO:, equilibrium is not obtained in less than 1 min, the generation of bicarbonate may be made rate limiting for the carboxylation of biotin. It was first demonstrated by Kaziro et al. (4) using pig heart propionyl-CoA carboxylase and lXO-labeled bicarbonate that, for each lx0 found incorporated in P,. two were found incorporated in the final product, methylmalonyl-CoA. These isotopelabeling studies, which also demonstrated HCO:, - and ADP-dependent ATP-““P, exchange and HCO:,-and Pi-dependent ATP-] ‘CJADP exchange, resulted in the proposal of a reaction mechanism for the carboxylation of biotin. This mechanism is
THE
CARBOXYLATION
mechaillustrated in Fig. la. Another nism, not unlike that in Fig. la but incorporating greater detail, is that of McClure et al. (6), which is illustrated in Fig. lb. This is a concerted mechanism whereby the biotin-HCOx~ reaction is concomitant with an ATP-HCO,,reaction. As pointed out by Kaziro et al., these results are also consistent with a stepwise mechanism involving the formation of an activated bicarbonate, perhaps carbonyl phosphate 0 II (HO-C-OPO,,‘-), which in turn carboxylates biotin. Other reaction mechanisms proposed include a stepwise mechanism whereby the biotin-bicarbonate reaction is preceded by phosphorylation of the enzyme. This mechanism is in accord with the experimental finding (7) that pyruvate carboxylase, purified from chicken liver mitochondria (unlike the acyl-CoA carboxylases), catalyzes a slow ATP- I ’ ‘C!IADP exchange which apparently proceeds without biotin participation; the investigators themselves point out that an abortive pathway is consistent with their results. Because of the ATP-:‘“P, and ATPATP + HCC$
OF
191
BIOTIN
I “CIADP exchanges and their dependencies, the stepwise mechanisms require an ordered release of Pi and ADP which may not precede the carboxylation of biotin. Finally, Bruice and Hegarty (8) have suggested the possibility that the initial attack on bicarbonate is by the ureido-carbony1 oxygen of biotin. In the course of the discussion which follows, the reaction mechanism outlined in Fig. la will be elaborated upon. STRUCTURAL RECOGNITION
INFORMATION ON OF BICARBONATE
Recently, the crystal structure of D-(+ )biotin has been determined (9) by the single crystal X-ray diffraction technique, affording a precise description of the molecular geometry of this coenzyme. A view of the packing and hydrogen bonding is given in Fig. 2a. Of particular interest in the crystal structure is the intermolecular hydrogen bonding between the two oxygen atoms of the carboxylic acid group of the valeric acid side chain and both the carbony1 oxygen and Nl’ of the ureido group of another molecule. The carboxylic acid functionality is not ionized so that one of
+ Enzyme-botn
R~tme ‘0 / P=O
0
-0’
FIG.
la.
Concerted
‘0 / PC0
!? HN’
BIOTIN
HN:
mechanism
of Kaziro
et al.
(4)
adon~n~
192
WILLIAM
C
STALLINGS
RI++
FIG.
lb.
More
detailed
concerted
mechanism
-0
/\
,o P=O
of McClure
et al.
(6).
FIG. 2a. The packing in the crystal structure of n-(+I-biotin is illustrated. Nl’ and 02’ of one molecule hydrogen bond with OlOA and OlOB (the carboxylic oxygens) of a neighboring molecule. (OlOA, hydrogen bound to 02’, is protonated.) OlOB is also the acceptor in an N3’ OlOB intermolecular hydrogen bond.
its oxygen atoms is covalently linked to a hydrogen atom and is thus capable of being a donor in a hydrogen bond with 02’ of the ureido ring. Biotin attaches CO, at
Nl’, attack by N3’ being hindered presumably for steric reasons involving the proximity of the valeric acid side chain. This scheme of hydrogen bonding is probably a
THE
CARBOXYLATION
5‘
OF
193
BIOTIN
16
FIG. 2b. Illustrated
are scale diagrams of structural data relevant to biotin recognition of bicarbonate. These were produced by calculating a least-squares plane through the ureido or urea carbon, nitrogen, and oxygen atoms. Deviations (in Angstrom units) from this plane are indicated when greater than 0.05 A. Hydrogen bond donor-acceptor distances are also included. (1) biotin; (2) oxybiotin; (3) dethiobiotin (in this case, complementary hydrogen bonding occurs between 02’ and N3’ of one molecule and the carboxylic acid functionality of a neighboring molecule); (4) urea-oxalic acid complex; (5) urea-phosphoric acid complex; (6) a relevant portion of the his-p-bromoanilide of CO, biotin which serves as a model of the reaction product of biotin and bicarbonate.
good model for the ureido recognition of bicarbonate since the carboxylic acid functionality in many ways resembles bicarbonate. Similar schemes of hydrogen
bonding are also possible and have been shown to exist in the recently determined crystal structures of dethiobiotin (10) and oxybiotin (11). The crystal structures of
194
WILLIAM
C. STALLINGS
urea complexed with oxalic acid (12) and phosphoric acid (13) also bear upon this point because of the similarity between urea and the ureido portion of biotin. In these structures, a similar 0. . . , NH.. .-. . .HO, . . .O hydrogen bonding pattern also occurs. Scale diagrams showing portions of the hydrogen bonding in each of these structures are presented in Fig. 2b. It would seem evident, then, that the ureido group possesses a geometry which is ideal for complementary hydrogen bonding with a protonated acid. This may be illustrated as
A model for biotin recognition of bicarbonate via hydrogen bonding is therefore proposed and this model is illustrated in Fig. 3. Relevant points include complementary hydrogen bonding between the ureido-carbony1 oxygen with the protonated oxygen of bicarbonate and Nl’ of the ureido group with an unprotonated oxygen of bicarbonate. This scheme of complementary hydrogen bonding is somewhat analogous to the complementarity in hydrogen bonding patterns observed in nucleic acids. A PROPOSED
MECHANISM ACTIVATION
OF
BIOTIN
This mode of biotin recognition of bicarbonate suggests a likely mechanism for the carboxylation reaction, [la]. The hydrogen bonded complex illustrated in Fig. 3 is probably stable, and it is the additional approach of ATP, providing chemical potential for the reaction, which triggers carboxylation at Nl’ by inducing the electronic rearrangements illustrated in Fig. 4; the mechanism presented here compares closely with that of Fig. la. In this mechanism, polarization of the car-bony1 bond, with corresponding enhanced nucleophilicity at Nl’ (the tautomerism of the ureido group, illustrated in Fig. 4b), results from hydrogen bonding with bicarbonate. This is a crucial step of the mechanism since, as has been pointed out (14), the nucleophilicity of Nl’ is prob-
FIG. 3. Proposed biotin-bicarbonate via hydrogen bonding.
recognition
ably very low, thus necessitating some form of biotin activation to enhance the nucleophilicity at this nitrogen. Hegarty et al. (151, having studied the reactivity of 2methoxy-2-imidazoline as a model for the enol form of biotin, found the nucleophilicity of this compound to be greatly enhanced relative to that of 2-imidazolidone, a model for the keto form of biotin. They therefore suggested that a probable form of activated biotin is the enol tautomer. It is perhaps interesting to note that, in the mechanism proposed here, tautomeric activation results from interaction with bicarbonate, the species with which biotin finally reacts. It is suggested that the activation consists of two proton transfers with resultant tautomerism of the ureido group. One proton is transferred from the protonated oxygen of bicarbonate to the ureido oxygen of biotin. Another proton is transfered from Nl’ of biotin to an oxygen of bicarbonate. Hence, the protons are transferred from their hydrogen bond donors to their respective hydrogen bond acceptors; the hydrogen bond donors of Fig. 4a become the hydrogen bond acceptors of Fig. 4b. The keto and enol tautomers, illustrated, respectively, in Figs. 4a and 4b, are probably in rapid equilibrium. The approach of ATP, indicated in Fig. 4b, shifts the equilibrium to the enol tautomer by reacting with the bicarbonate oxygen which is hydrogen-bonded to Nl’ of biotin; as indicated in Figs. 4b and 4c, this triggers the completion of the reaction. In the crystal structure of the urea-phosphoric acid com-
FIG. 4. Proposed reaction mechanism. Although a concerted reaction mechanism is suggested, it is illustrated in three parts for clarity. (ai Recognition of biotin by bicarbonate. Hydrogen bonding of bicarbonate to carbonyl oxygen and Nl’ of biotin as shown by the data discussed in this paper. tb) Tautomerism as a result of hydrogen bonding. Polarization of the carbonyl group and increased nucleophilicity at Nl’. ATP participation at this point shifts the equilibrium to the enol tautomer by reacting with the bicarbonate oxygen which is involved in the hydrogen bond with Nl’ of biotin. The ATP conformation illustrated has been observed crystallographically (26). (c) Covalent bond formation between Nl’ of biotin and C of bicarbonate. Formation of P, and ADP from ATP.
196
WILLIAM
C. STALLINGS TABLE
GEOMETRIC
~.~-.~ Uranium
DATA Substance
RELEVANT
TO
THE POLARIZATION Reference
~ nitrate
I AND TAUTOMERISM Has the oxygen been protonated?
OF c-o
THE
(A)
16
Yes
1.298 (1)
17
Yes
1.301 (4)
13
Partially
1.281 (3,
12
No
1.261 (3)
Urea
21
NO
1.270 (7)
N-Methylurea
22
No
1.248 (1)
9
No
1.249
(6)
23
No
1.236
(6)
Dethiobiotin
10
No
1.244
(4)
Oxybiotin
11
No
1.270
23
No
1.243
N-Methyluronium Urea-phosphoric Urea-oxalic
nitrate acid
complex
acid complex
Biotin Biotin
Azabiotin
methyl
ester
hydrochloride
hydrate
UREIDO
GROUP" C-N
(11
1.312 1.315 1.309 1.307 1.324 1.323 1.329 1.329 1.326 1.326 1.340 1.336 1.332 1.351 1.350 1.351 1.346 1.346 1.328 1.290 1.345 1.348 1.37
(1) (1) (41 (4) (2) (2) (4) (6, (6) (6) (2, (2) (6, (71 (7) (6) (4) (41
12 barbiturate structures averaged 24 No 1.21 -~~-~~ ” This table indicates that, in general, a tautomeric-like geometry is observed upon ureido-oxygen protonation. It further illustrates that in biotin, as in urea, the polarized (tautomeric) forms of their respective resomers contribute significantly to the overall electronic structures. The polarized forms, however, do not always contribute as significantly in a ureido system since a recent survey (24) of 12 barbiturate structures (barbiturates contain a ureido-like chemical unit) indicates the presence of unpolarized carbonyl C=O bonds. It is concluded, then, that biotin and urea are similar in their polarizability but that not all molecules containing the ureido group have the ability to be similarly polarized. This has been discussed more fully elsewhere (9, IO). For comparison, reference C-O, C=O, C-N, and C=N bond lengths are quoted (25): C=O, 1.21 A; C-O, 1.43 A; C=N, 1.25 A; C-N, 1.47 A.
plex’ (13), the hydrogen atom involved in the hydrogen bond between the urea carbony1 functionality and a protonated oxygen of H,PO, is actually equidistant (1.223 and 1.207 A) from these two oxygen atoms, indicative of the possibility (see Fig. 4b) of protonation of the ureido oxygen in biotin as a result of the proposed hydrogen bonding scheme with bicarbonate. In the crystal structure of uronium nitrate (see Footnote 1) (16) and N-methyluronium nitrate (171, the proton has actually been transferred to the carbonyl oxygen although ’ These structures have been determined by the technique of neutron diffraction such that the hydrogen atoms have been located with precision not obtainable using the X-ray technique.
complementary hydrogen bonding (as described) is not observed. Changes which occur, upon protonation, in the C-O and C-N bond lengths are significant, indicating extensive polarization of the C-O and double-bond character in the C-N bond, the electronic rearrangement suggested in Fig. 4b and triggered by the proposed proton transfer from bicarbonate to ureido oxygen. Geometric data relevant to the polarization and the tautomerism of the ureido group are summarized in Table I. The result of increased nucleophilicity at Nl’ is covalent bond formation between this nitrogen and the carbon atom of bicarbonate. Concomitantly, the protonated ox-
THE
CARBOXYLATION
ygen of bicarbonate is added to ATP which is cleaved to ADP and Pi. This is illustrated in Fig. 4~. The crystal structure of the bis-p-bromoanilide of carbon dioxide biotin (18) perhaps provides a good model for the final product. The molecular formula of this compound is illustrated below and a scale diagram of its structure has been included in Fig. 2b. O-----H \
II HN
&
P\
i
43
!3r
“-No
s
“*a a
It is interesting to note that the crystal structure contains an intramolecular N. ’ .O hydrogen bond (2.64 A1 between the ureido oxygen and the protonated nitrogen atom of the group attached to Nl’. An intramolecular 0. .O hydrogen bond has therefore been incorporated into Fig. 4c. Although this mechanism does not account for the role of the divalent metal ion, it is not difficult to speculate about its possible function. This might be one of coordination involving the sulfur atom or, perhaps even more likely, it may serve to properly coordinate the triphosphate portion of ATP and bicarbonate in a geometry which induces the carboxylation reaction. This latter idea has been incorporated into the rather detailed mechanism of McClure et al. (6). The mechanism also does not provide a description of the interaction and reaction between bicarbonate and ATP. (It is assumed that ATP is not protonated in this reaction. Otherwise, the ureido group might also be a recognition site for this portion of ATP, especially in view of the existence of a tightly bound urea-triprotonated phosphoric acid complex.) Structural models derived from experimental data are lacking for these cases.
OF
BIOTIN
197
By superimposing the bicyclic ring systems of biotin and the bis-p-bromoanilide of CO,-biotin as illustrated in Fig. 2b, a sense of the dynamics of the reaction mechanism is achieved. This is illustrated in Fig. 5. Bicarbonate must undergo a relative rotation and translation toward Nl’ to accomplish covalent bond formation. The proposed mechanism is in accord with the kinetic isotope exchange data of Kaziro et al. (4), mentioned earlier. Figure 4 illustrates a concerted reaction mechanism involving concomitant biotin-HCO,, and HCO,,--ATP reactions; these reactions are preceded by biotin-HCO,; complexation. A stepwise mechanism involving prior formation of an activated bicarbon-
ate such as HO-C-OPO,,” cannot be excluded on the basis of the arguments presented here since this form of bicarbonate would be capable of similar complementary hydrogen bonding with biotin. Recent studies (19) of sheep kidney pyruvate carboxylase have provided evidence against a stepwise mechanism involving phosphorylation of the enzyme prior to the carboxylation reaction. This mechanism is also in
FIG. 5. Dynamic representation of the reaction mechanism obtained by superimposing the bicyclic ring system of biotin and the bis-p-bromoanilide of CO, biotin as illustrated in Fig. 2b. This diagram illustrates the reactants (biotin and bicarbonate), a model of their reaction product, and the relative dynamics of the proposed reaction mechanism. It is evident that to accomplish covalent bond formation between Nl’ of biotin and C of bicarbonate, hydrogen bound bicarbonate must undergo a relative rotation, indicated by the arrow, and translation toward Nl’. The initial position of bicarbonate is indicated by the letters a, b, c, and d. After covalent bond formation, a, b, and c have moved to a’, b’, and c’ with cleavage of b-d bond; d is transferred to ATP (as d’).
198
WILLIAM
C. STALLINGS
accord with some finer details observed by various investigators. Caplow (14), using 2-imidazolidone as a model for biotin, was unable to demonstrate any reactivity between this compound and such carboxylating agents as p-nitrophenyl acetate, acetylimidazole, and acetyl-3-methylimidazolium chloride. He concluded that, in biotin, the nucleophilicity at Nl’ is probably very low and that, in addition to the possibility of bicarbonate activation, the nucleophilicity at Nl’ must be increased by some form of biotin activation. Note also that the enol form of biotin, which has been suggested as the probable intermediate (15), is involved in the mechanism proposed here. A probable mechanism of activation, suggested by Gressier et al. (20), is via polarization of the ureido-carbonyl group by hydrogen bonding. Such a scheme would stabilize the polarized forms of the biotin resomers and increase the
presumably reacts. The tautomerism of biotin which results from this hydrogen bonding increases the nucleophilicity of Nl’, and the participation of ATP provides chemical potential, driving the reaction to completion. In this way, a covalent bond is formed between Nl’ and the carbon atom of bicarbonate. At the same time, oxygen is added to ATP resulting in its cleavage to ADP and P,. This mechanism represents an example of substrate activation; the binding of bicarbonate, the substrate in question, increases the nucleophilicity of biotin. It is possible that this mechanism is essentially more general. For example, the reaction between citrulline and aspartic acid, catalyzed by argininosuccinate synthetase, may proceed by a similar mechanistic pathway and with a similar pattern of recognition. The reaction also involves ATP. This question is currently being investigated. ACKNOWLEDGMENTS
acidity of the Nl’ proton, leading to enhanced nucleophilicity at Nl’. The recent single crystal studies of biotin (9, 10) have revealed that the carbonyl oxygen is capable of forming a strong hydrogen bond, and an analysis of the bond lengths indicates that the polarized forms of the biotin resomers contribute significantly to the overall electronic structure. SUMMARY
In summary, then, a probable mode of recognition between the ureido portion of biotin and bicarbonate is via a scheme of complementary hydrogen bonding involving the carbonyl group and Nl’ of biotin. This mode of recognition has suggested a mechanism which explains nucleophilic activation at Nl’ of biotin. This activation is via polarization of the ureido-carbonyl group as a result of hydrogen bonding with bicarbonate, the species with which biotin
I would like to thank Dr. Mary Wimmer for helpful conversations. I would like to acknowledge enlightening conversations with Dr. George DeTitta and to thank both him and Dr. R. Parthasarathy for giving me the oxybiotin coordinates prior to publication. I would also especially like to thank Dr. Jenny Glusker for making many valuable suggestions regarding both the form and the content of this paper as well as for the preparation of its figures. This research was supported by USPHS Grants F32 CA-05322, CA-10925, CA-06927, and RR-05539 from the National Institutes of Health, by AG-370 from the National Science Foundation, and by an appropriation from the Commonwealth of Pennsylvania. REFERENCES 1. Moss, J., AND zymol. Relat. 2. (a) GUCHHAIT,
M. D. 11971) Advan. EnAreas Mol. Bid. 35, 321-442. R. B., POLAKIS, S. E., DIMROTH, P., STOLL, E., Moss, J., AND LANE, M. D. (1974) J. Biol. Chem. 249, 6633-6645; (b) GUCHHAIT, R. B., POLAKIS, S. E., HOLLIS, D., FENSELAU, C., AND LANE, M. D. (197415. Bid. Chem. 249, 6646-6656; (cl POLAKIS, S. E., GUCHHAIT, R. B., ZWEIGEL, E. E., AND LANE, M. D. 11974)J. Biol. Chem. 249, 6657-6667. 3. FUNG, C. H., GUPTA, R. K., AND MILDVAN, A. S. (1976) Biochemistry 15, 85-92. 4. KAZIRO, OCHOA,
LANE,
Y., HASS, S. (1962)
L.
F.,
J. Bid.
BOYER,
Chem.
P.
D.,
237,
AND
1460-
THE
CARBOXYLATION
1468. 5. FITMORE, D. L., AND COOPER, T. G. (1970) d. Theor. Biol. 29, 131-145. 6. MCCLURE, W. R., LARDY, H. A., AND CLELAND, W. W. (1971)J. Biol. Chem. 246, 3584-3590. 7. SCRUTTON, M. C., AND UTTER, M. F. (1965) J. Biol. Chem. 240, 3714-3723. 8. BRUICE, T. C., AND HEGARTY, A. F. (1970) Proc. Nat. Acad. Sci. USA 65, 805-809. 9. DETITTA, G. T., EDMONDS, J. W., STALLING, W., AND DONOHUE, J. (1976) J. Amer. Chem. Sot. 98, 1920-1926. 10. CHEN, C. S., PARTHASARATHY, R., AND DETITTA, G. T. (1976) J. Amer. Chem. Sot. 98, 49834990. 11. DETITTA, G. T., AND PARTHASARATHY, R. (1976) Unpublished results. 12. HARKEMA, S., BATS, J. W., WEYENBERG, A. M., AND FEIL, D. (1972) Acta Crystallogr. B28, 1646-1648; see also: HARKEMA, S., BATS, J. W., WEYENBERG, A. M., AND FEIL, D. (19731 Acta Crystallogr. B29, 143. 13. KOSTANSEK, E. C., AND BUSING, W. R. (1972) Acta Crystallogr. R28, 2454-2459. 14. CAPLOW, M. (1965) J. Amer. Chem. Sot. 87, 5774-5785. 15. HEGARTY, A. F., BRUICE, T. F., AND BENKOVIC. S. J. (1969) Chem. Commun. 1173-1174. 16. WORSHAM, J. E., AND BUSING, W. R. (1969)Acta Crystallogr. B25, 572-578.
OF
BIOTIN
199
17. SELMAN, W. F., AND HARKEMA, S. (1971) Progress Report, Chemical Physics Laboratory, Twente University of Technology. 18. BONNEMERE, C., HAMILTON, J. A., STEINRAUF. L. K., AND KNAPPE, J. (1965) Biochemistry 4. 240-245. 19. ASHMAN, L. K., AND KEECH, D. B. (1975)J. Bzol. Chem. 250. 14-21. 20. GRESSIER, R., SIGEL, H., WRIGHT, L. D., AND MCCORMICK, D. B. (1973) Biochemistry 12, 1917-1922. 21. CARON, A., AND DONOHUE, J. (19691 Acta Crystallogr. B25, 404. 22. HUISZOON, C., AND TIEMESSEN, G. W. H. (1976) Acta Crystallogr. B32, 160441606. 23. DETITTA, G. T., GLICK, M., PARTHASARATHY, R., AND STALLINGS, W. (1976) Presented at the American Crystallographic Association Meeting, Northwestern University, Evanston, Ill., Aug. 9-12, 1976, Abstracts. 73-74. 24. CRAVEN, B. M., CUSATIS, C., GARLAND, G. L., AND VIZZINI, E. A. (1973) J. Mol. Struct. 16, 331-342. 25. PAULING, L. (1960) in The Nature of the Chemical Bond, pp. 228-229, Cornell University Press, Ithaca. 26. KENNARD, O., ISAACS, N. W., MOTHERWELL, W. D. S., COPPOLA, J. C., WAMPLER. D. L., LARSON, A. C.. AND WATSON, D. G. (1971) Proc. Roy. Sot. hndon SeF. A 325, 401-436.