From the European Molecular Biology Laboratory, c/o
DESY, Notkestrasse 85, 22603 Hamburg, Germany, ¶ Fachrichtung
12.4, Biochemie, Postfach 151150, Universitaet des Saarlandes, 66041 Saarbruecken, Germany,
Instituto di Scienze Biochimiche,
Universita di Parma, viale delle Scienze, 43000 Parma, Italy, and
** Biokemi, Kemicentrum, Lunds Universitet, Box 124, 22100 Lund, Sweden
Received for publication, December 1, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Atomic (1 Å) resolution x-ray structures of
horse liver alcohol dehydrogenase in complex with NADH revealed
the formation of an adduct in the active site between a metal-bound
water and NADH. Furthermore, a pronounced distortion of the pyridine
ring of NADH was observed. A series of quantum chemical calculations on
the water-nicotinamide adduct showed that the puckering of the pyridine
ring in the crystal structures can only be reproduced when the water is
considered a hydroxide ion. These observations provide fundamental
insight into the enzymatic activation of NADH for hydride transfer.
Nicotinamide adenine dinucleotide NAD(H) is the most abundant
electron carrier in cell metabolism. It exists in an oxidized (NAD+) and a reduced (NADH) form, and both species are
stable under physiological conditions. Its capacity as a redox agent is
exploited by numerous enzymes that catalyze reactions in which
NAD+ is reduced to NADH and vice versa. The
interconversion of NAD+ and NADH is achieved by the
transfer of a hydride ion (two electrons and a proton) between a
substrate and NAD(H). A study on deuterated model compounds has shown
that the hydride carrier is the C-4 atom on the pyridine ring of
the nicotinamide and that the transfer process is stereospecific (Ref.
1 and Fig. 1). Theoretical calculations
on the nicotinamide indicated that deformation of the pyridine ring
into a boat conformation enhances hydride transfer (2). It has been
observed by UV-visible spectroscopy and NMR that the puckering of the
pyridine ring changes upon enzyme binding (3). Until now it was unclear
how an enzyme could steer the puckering of the ring to facilitate
hydride transfer. Here, we present for the first time an example of
enzymatic activation of NADH as it was found in a complex of NADH with
horse liver alcohol dehydrogenase (LADH;
EC.1.1.1.1).1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 1.
a, definition of the puckering of the
nicotinamide moiety. Hydride transfer takes place at the C-4 atom of
the pyridine ring and is stereospecific. In LADH, the HR
hydrogen is exchanged. XN describes the dihedral between
the ribose and the nicotinamide moiety. The dihedral XAM
describes the orientation of the carboxamide in relation to the
nicotinamide ring. The puckering of the ring is described by
N accounting for the pyramidalization of N-1. It is the
angle between the planes of N-1[-]C-2[-]C-6 and
C-2[-]C-3[-]C-6. The tendency for C-4 to be out of plane is
represented by the angle
C between the planes of
C-3[-]C-4[-]C-5 and C-2[-]C-3[-]C-6. Twist T displays the
distortion in the boat form, calculated from the distance between C-5
and the plane formed by C-2[-]C-3[-]C-6. b, the
hydrogen bonding network between the nicotinamide moiety and the enzyme
as observed in the Zn-MPD-NADH-LADH crystal structure. The fragments
used in the quantum chemical calculations to mimic this environment are
given in parentheses.
LADH is an NAD(H)-dependent enzyme that catalyzes the oxidation of primary and secondary alcohols to aldehydes (4). It belongs to the Class I of the alcohol dehydrogenase family (5), which also contains yeast alcohol dehydrogenase and the human zinc-dependent alcohol dehydrogenase. The yeast enzyme is the key mediator of alcohol production, whereas the human enzyme cleanses the blood from poisonous compounds. Other functions that have been assigned to the human enzyme are the involvement in the hormonal household by switching on and off steroids like testosterone. It has been associated with the production and neutralization of free radicals, thereby protecting the organism against DNA damage (6). Finally, alcohol dehydrogenase is thought to play a role in the development of the organism through retinoid regulation (7). Retinoids are vitamin A derivatives that regulate the expression of various genes involved in embryonic growth. It has been observed that heavy drinking leads to retarded growth and night blindness. This can be explained by the fact that the continuous oxidation of ethanol interferes with the reduction of carotene to vitamin A.
LADH is a homodimer with 374 residues and two zinc sites in each monomer. One zinc is thought to play a structural role, and the other forms the core of the active site. The latter is liganded to two cysteine residues (Cys46 and Cys174) and a histidine (His67). The x-ray structure of the apo-enzyme indicated that the fourth metal ligand was a water molecule (4). The enzyme has been the focus of intensive studies involving kinetics, x-ray crystallography, and spectroscopic methods (8). Much is known about the kinetics of the assembly of the enzyme-cofactor complex (9), but the actual mechanism for hydride transfer and proton release is still under debate (10).
Alternative reaction mechanisms have been proposed for LADH. In one of
these (4), LADH is regarded an exception among
zinc-dependent enzymes in that no water molecule is
involved in the reaction. This is the standard textbook description of
the LADH mechanism (11). In the second mechanism (12), the water
molecule is linked to the zinc ion during the reaction and goes through
a cycle of (de)protonation. This mechanism demands a five coordination around the metal. Such coordination of the metal is thought to be
highly improbable, because a fifth ligand would cause collisions with
surrounding residues (4). EPR (13) and NMR (14) data on a
cobalt-substituted enzyme and perturbed angular correlation of
-rays measurements (15) on cadmium-substituted enzyme
indicate the existence of a five coordinate intermediate.
Unbound NADH displays an absorption maximum at 340 nm in the UV-visible range. When NADH is bound to LADH, the maximum shifts to 325 nm. Early on, it was proposed that this blue shift is caused by the formation of a bond between the catalytic zinc ion and the nicotinamide (16). This proposal was discarded after elucidation of the 3 Å resolution x-ray structure of the NADH-LADH complex because it was concluded that the distance between the zinc ion and the nicotinamide was too long.
To access the minute structural changes that an enzymatic environment
might impose on NAD(H), it is necessary to collect structural data at a
truly atomic level. Atomic resolution x-ray data provide an accuracy in
atomic positions in the range of 0.03 Å (17, 18), and detailed
features of the active site of the protein structure become visible
that are lost at lower resolution. Once the precise positions of all
the atoms that are essential for the reaction to proceed are known,
detailed theoretical studies can be launched to unravel the reaction mechanism.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sample Preparation, X-ray Data Collection, and Refinement-- EE-isozyme of LADH was prepared according to procedures described in Ref. 19. Data were collected on native enzyme in complex with 2-methyl 2,4-pentanediol (MPD) and NADH further referred to as the Zn-MPD-NADH-LADH complex. The zinc was replaced by cadmium following protocols described in Refs. 13 and 20. A cadmium-substituted enzyme was complexed with MPD and NADH and is further referred to as the Cd-MPD-NADH-LADH complex. The Zn-MPD-NADH-LADH complex was crystallized with polyethylene glycol 400 at a concentration of 20% in 0.05 M Tris-HCl buffer at pH 8.2 with 0.5% MPD present. The Cd-MPD-NADH-LADH complex was crystallized by dialysis against 50 mM Tris-HCl, pH 8.2, and stepwise addition of MPD up to a concentration of 12%. Subsequently, polyethylene glycol 400 was added as a cryoprotectant to a final concentration of 20%. Diffraction data were collected on the BW7B beamline at the EMBL Outstation Hamburg, DESY, from flash-cooled crystals, using an image plate from MAR X-ray Research GmbH (Hamburg, Germany). An additional data set was collected on the Cd-MPD-NADH-LADH complex at 277 K. Data were processed, merged, and scaled with the HKL suite (21). Statistics are summarized in Table I.
Rfree was used to cross-validate the refinement protocol. The "free" reflections were included in the final refinement round. The Zn-Me2SO-NADH-LADH structure (Protein Data Bank reference code 2OHX; Ref. 22) determined to a resolution of 1.8 Å was used as an initial model for refinement. The 1 Å resolution structures were refined with SHELX-97 (23) and REFMAC (24) using anisotropic displacement parameters and H atoms at idealized positions. The resolution of the x-ray data allowed the geometry of the nicotinamide of NADH to be freed from restraints to get an accurate picture of the puckering of the pyridine ring. No restraints were applied either to metal-ligand distances. The Cd-MPD-LADH-NADH complex collected at 277 K was refined using the cryo structure as a starting model. Because of the low number of observations, it was not possible to release the restraints in the active site in this case. X-ray structure factor amplitudes and the derived atoms coordinates have been deposited in the Protein Data Bank under accession numbers R1HEUSF, 1HEU, R1HF3SF, 1HF3, R1HETSF, and 1HET.
UV-visible Spectroscopy on the Crystals-- Single crystal polarized absorption spectra were recorded at 283 K using a Zeiss MpM800 microspectrophotometer on a crystal placed in a quartz flow cell with the incident beam perpendicular to the face of the crystal (25). The crystal was oriented with either of the two principle optical directions parallel to the electric vector of the polarized light.
Theoretical Calculations-- The nicotinamide moiety was truncated in such a way that the oxygen connecting the ribose to the pyrophosphate group was replaced by a hydrogen. Starting coordinates for this nicotinamide moiety were taken from the Zn-MPD-NADH-LADH crystal complex, and the geometry was optimized at the Hartree-Fock level using the GAMESS (26) software package with a 6-31+G** basis set. The hydrogen bonds that influence the geometry of the nicotinamide moiety were initially mimicked by fixing the dihedral angles XN and XAM (Fig. 1a). A water molecule was placed at 1.7 Å from C-6 of the pyridine ring of the nicotinamide. The geometry of the nicotinamide moiety was optimized to fit the new conditions with constraints applied to the oxygen of the water molecule to keep the water in a fixed position relative to the pyridine ring as observed in the Zn-MPD-NADH-LADH x-ray structure.
For the reduced nicotinamide system these calculations were then
extended to further approximate the electronic environment of the
nicotinamide moiety by the introduction of precalculated potentials
placed at all hydrogen bonding positions. Fig. 1b shows the
enzyme hydrogen bonding environment in the crystal structure and the
fragments used for the electronic potential calculations. Their
potential, dipole, and quadropole moments were incorporated at fixed
positions. Finally, the zinc atom was included in a similar manner
together with the two SH groups and an ammonia mimicking its cysteine
and histidine ligands in the crystal structure.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Active Site Interactions-- X-ray data were collected to a resolution of 1 Å on native zinc LADH and the cadmium-substituted enzyme in complex with NADH and MPD (Table I). MPD was used as a precipitating agent during crystallization of the cadmium-substituted protein but only as a minor addition in case of the zinc enzyme. MPD formed an abortive complex in both cases. Two complexes were measured under cryogenic conditions. A separate data set was collected on the Cd-MPD-NADH-LADH complex at 277 K to a resolution of 1.95 Å that has shown that there were no gross structural changes in the active site induced by flash cooling.
|
In both subunits of the Zn-MPD-NADH-LADH complex there is a residual
density peak close to the C-6 atom of the nicotinamide (W1 in Fig.
2a and Table
II). This peak is within inner
coordination sphere distance of the catalytic zinc ion. Another peak of
similar height and distance from the metal (W2) is also seen in both
monomers. The second location corresponds to the position otherwise
taken by a zinc-interating atom of a substrate (4). The distance between the two peaks is 1.47 Å. In the Cd-MPD-NADH-LADH complex, the
distances are essentially the same (Table II). In both monomers and in
both complexes the W2 site is remote from the C-6 NADH atom. Similar
peaks are observed in the active site of the Cd-MPD-NADH-LADH complex
measured at 277 K. Accurate distances cannot be obtained from this
structure as a result of the limited resolution.
|
|
The proton shuffling residue Ser48 is equidistant to both peaks in all complex structures. In each case, the pyridine ring of NADH is puckered in a twisted boat conformation. We interpret the two peaks together to represent a metal-bound water molecule occupying two alternative positions, partially forming an adduct with the pyridine ring of NADH.
Substrate Conversion--
UV-visible spectra were recorded on
crystals from the same batch as those used for x-ray data collection.
Fig. 3 shows an absorption maximum at 325 nm typical for enzyme bound NADH. Subsequent treatment with substrates
shows that the crystalline enzyme is active. This allows us to
interpret the crystal structures in terms of the enzymatic reaction
mechanism.
|
NADH Adducts-- Water involvement in (de)protonation reactions in LADH has played a central role in explaining pH dependences for enzymatic activity (27). In many other zinc enzymes (28), the metal-bound water ought to be deprotonated to facilitate catalysis. Because of the fact that the water is disordered, it is not possible to assign its protonation state directly from the x-ray structure. To establish whether the adduct observed involves plain water or a hydroxide ion, quantum chemical calculations were performed on a nicotinamide model system (Fig. 1b).
Our calculations indicate that the pyridine ring structure is a highly
sensitive system in terms of basis set and of its electronic environment. The system was initially modeled by fixing the two dihedral angles XN and XAM (Fig. 1a)
as described under `Experimental Procedures` and presented in Tables
III and
IV. The unperturbed oxidized
nicotinamide (NIC+) and its adducts with H2O
and OH are essentially planar. The unperturbed reduced
nicotinamide (NICH) shows some deviations from planarity. Pronounced
puckering is seen in both NICH adducts. It can be seen in Fig.
4 that the NICH-OH
mostly
resembles the puckering observed in the pyridine rings of the crystal
structures. Furthermore, an increase in bond length for C-5[en]C-6 is
observed in the reduced NICH adducts. In the calculated structure of
the unperturbed reduced nicotinamide, the bond length is 1.33 Å corresponding to a double bond (Fig. 2b). In the calculated
structure of the reduced OH
adduct, the bond length is
1.46 Å (Fig. 2c). In the atomic resolution crystal
structures, the C-5[en]C-6 bond length is 1.41 Å.
|
|
|
For the NADH-OH adduct fixation of the two dihedral
angles proved to be a valid approximation. The geometry of the system did not change when the constraints on the dihedral angles
XN and XAM were released and hydrogen bonding
network was introduced in the form of fragments. For the
H2O adduct, the removal of the dihedral angle constraints
resulted in significant deviations from the crystal structure. This is
a possible indication that an NADH-H2O adduct is unlikely
to acquire the observed crystal structure geometry. The introduction of
zinc to the NICH-OH
system affected mainly the charge
distribution, and the electronegativity of the HR hydrogen became more profound.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because the crystal structures considered here all contain NADH, we restrict ourselves to describe the implications of adduct formation for NADH oxidation and aldehyde reduction. The forward reaction will be ruled by the same mechanism of adduct formation, but NAD+ might be oriented toward the water molecule in a different manner. No crystal structure of a binary LADH-NAD+ complex is available so far.
In the reduced OH ring adduct, the ProR
hydrogen at the C-4 atom is in a pseudo-axial position. The bonding
order of the C-4[-]HR bond decreases indicating a
tendency for the ProR to leave according to the
stereospecific requirements (Table IV). Based on the results of the
calculations and comparison with the crystal structures, we suggest
that OH
adduct formation is part of the activation
process for hydride transfer from the reduced cofactor in the LADH-NADH
complex. The hydrogen bonding between the nicotinamide moiety and the
protein plays mainly a structural role of holding NADH in place.
In Fig. 5 a revised reaction mechanism is
proposed that is an extension of the `dissenting` mechanism that
incorporates the metal-bound water (12). It was expected that a five
coordination of the metal leads to collisions with surrounding residues
or with the cofactor NAD(H) (4). As can be seen in the crystal structures presented here such a `collision` is indeed observed. The
revision of the mechanism alludes to the functional displacement of the
water and the formation of a nicotinamide derivative. The metal-bound
water alternates between two positions. The movement of the
water/OH is directed at the C-6 of the nicotinamide
guided by Ser48. The formation of a nicotinamide derivative
leads to a redistribution of electric charge along the ring, inducing
hydride transfer. The (de)protonation of the alcohol goes through the
metal-bound water. In this manner, the enzyme can influence the course
of the reaction by changing the coordination chemistry of the metal and
the proton flow toward the active site.
|
The new reaction mechanism resolves controversy over spectroscopic data
on the metal coordination during the reaction. Perturbed angular
correlation of -rays measurements on the binary LADH-NADH complex
revealed a mixed geometry of equal occupation (29). This would imply
that already in the binary complex, water displacement and adduct
formation occurs. Large shifts are observed in the metal spectra of
cobalt-substituted (30) and copper-substituted (8) enzymes upon NADH
binding. These shifts can now be attributed to geometrical changes in
the metal coordination sphere.
The presence of a water molecule in the vicinity of the nicotinamide has been found in several other protein structures. In a complex of dihydrofolate reductase, folate, and NAD+ at a resolution of 1.6 Å (31), a water is situated at 3.2 Å of C2 of the pyridine ring. In a complex of ferredoxin reductase with NADP+ and FAD (32), a close interaction between a protein residue (Thr166) and the C-6 (3.2 Å) of the pyridine ring is found. The distances quoted here are from x-ray structures with a restrained geometry, which implies that all the atoms that are expected to be nonbonding are kept at a distance that is in agreement with a van der Waal's radius of 3.3 Å. Given sufficiently high resolution of the x-ray data, the release of such restraints might reveal other enzyme-coenzyme complex structures with close interactions between the pyridine ring and neighboring oxygen atoms.
The work presented here highlights an essential aspect of the
NAD(P)-dependent dehydrogenation: the enzymatic activation
of the cofactor. The combination of truly atomic resolution x-ray crystallography and quantum chemical calculations provided the opportunity to study enzymatic catalysis in unprecedented detail and
enabled us to detect that NADH is indirectly linked to the catalytic
metal ion. This observation is independent of the nature of the
transition metal and the temperature of the environment. We propose
that the link established by the enzyme activates the NAD(H) molecule
to become involved in hydride transfer. Further atomic resolution x-ray
studies and extended theoretical calculations on other dehydrogenases
will certainly lead to a more complete understanding of
NAD(P)-dependent hydride transfer.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Z. Dauter for help in data collection and Rector Inge Jonsson and Dr. Jan Lundgren at Stockholm University for constant support. We thank the Associated Editor and referees for constructive critique.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the European Commission on Biotechnology Research and Technological Development Grant BIO4-CT96-0189.
** To whom correspondence should be addressed: EMBL c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany. Tel.: 49-40-89902121; Fax: 49-40-89902149; E-mail: victor@embl-hamburg.de.
§§ Supported by European Commission on Training and Mobility of Researchers/Programme, Access to Large-Scale Facilities Grant ERBFMGECT980134.
Published, JBC Papers in Press, December 28, 2000, DOI 10.1074/jbc.M010870200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LADH, liver alcohol dehydrogenase; MPD, 2-methyl 2,4-pentanediol; NICH, reduced nicotinamide.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Loewus, F. A., Westheimer, F. H., and Vennesland, B. (1953) J. Am. Chem. Soc. 75, 5018-5023 |
2. | Almarsson, O., and Bruice, T. C. (1993) J. Am. Chem. Soc. 115, 2125-2138 |
3. | Burke, J. R., and Frey, P. A. (1993) Biochemistry 32, 13220-13230[Medline] [Order article via Infotrieve] |
4. | Eklund, H., and Brändén, C. I. (1987) in Biological Macromolecules and Assemblies (Jurnak, F. A. , and McPherson, A., eds) , pp. 73-143, John Wiley & Sons, New York |
5. | Jörnvall, H., Persson, B., and Jeffrey, J. (1987) Eur. J. Biochem. 167, 195-201[Abstract] |
6. | Mantle, D., and Preedy, V. R. (1999) Adverse Drug React. Toxicol. Rev. 18, 235-252[Medline] [Order article via Infotrieve] |
7. | Foglio, M. H., and Duester, G. (1999) Biochim. Biophys. Acta 1432, 239-250[Medline] [Order article via Infotrieve] |
8. | Bertini, I., Luchinat, C., Maret, W., and Zeppezauer, M. (eds) (1986) Zinc Enzymes , pp. 377-492, Birkhäuser, Stuttgart |
9. | Adolph, H. W., Kiefer, M., and Cedergren-Zeppezauer, E. S. (1997) Biochemistry 36, 8743-8754[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Northrup, D. B.,
and Cho, Y.-K.
(2000)
Biophys. J.
79,
1621-1628 |
11. | Fersht, A. (1999) Structure and Mechanism in Protein Science , pp. 460-465, W. H. Freeman and Company, New York |
12. | Dworschack, R. T., and Plapp, B. V. (1977) Biochemistry 16, 111-116[Medline] [Order article via Infotrieve] |
13. | Maret, W. (1989) Biochemistry 28, 9944-9949[Medline] [Order article via Infotrieve] |
14. | Sloan, D. L., Young, J. M., and Mildvan, A. S. (1975) Biochemistry 14, 1998-2008[Medline] [Order article via Infotrieve] |
15. | Hemmingsen, L., Bauer, R., Bjerrum, M. J., Zeppezauer, M., Adolph, H. W., Formicka, G., and Cedergren-Zeppezauer, E. S. (1995) Biochemistry 34, 7145-7153[Medline] [Order article via Infotrieve] |
16. | Mahler, H. R., and Douglas, J. J. (1957) J. Am. Chem. Soc. 79, 1159-1166 |
17. | Dauter, Z., Lamzin, V. S., and Wilson, K. S. (1997) Curr. Opin. Struct. Biol. 7, 681-688[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Kachalova, G. S.,
Popov, A. N.,
and Bartunik, H. D.
(1999)
Science
284,
473-476 |
19. | Hubatsch, I., Maurer, P., Engel, D., and Adolph, H. W. (1995) J. Chromatogr. 711, 105-112[CrossRef] |
20. | Maret, W., Andersson, I., Dietrich, H., Schneider-Bernlöhr, H., Einarsson, R., and Zeppezauer, M. (1979) Eur. J. Biochem. 98, 501-512[Abstract] |
21. | Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326 |
22. | Al-Karadaghi, S., Cedergren-Zeppezauer, E. S., Hovmöller, S., Petratos, K., Dauter, Z., and Wilson, K. S. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 793-807[CrossRef] |
23. | Sheldrick, G. M., and Schneider, T. R. (1997) Methods Enzymol. 276, 319-343[CrossRef] |
24. | Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S., and Dodson, E. J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 247-255[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Merli, A.,
Brodersen, D. E.,
Morini, B.,
Chen, Z.,
Durley, R. C. E.,
Matthews, F. S.,
Davidson, V. L.,
and Rossi, G. L.
(1996)
J. Biol. Chem.
271,
9177-9180 |
26. | Schmidt, M. W., Baldridge, K. K., Boatz, J. A., Elbert, S. T., Gordon, M. S., Jensen, J. H., Koseki, S., Matsumaga, N., Nguyen, K. A., Su, S. J., Windus, T. L., Dupuis, M., and Montgomery, J. A. (1993) J. Comput. Chem. 14, 1347-1363 |
27. | Kvassman, J., and Pettersson, G. (1980) Eur. J. Biochem. 103, 565-575[Abstract] |
28. | Coleman, J. E. (1998) Curr. Opin. Chem. Biol. 2, 222-234[CrossRef][Medline] [Order article via Infotrieve] |
29. | Hemmingsen, L., Bauer, R., Bjerrum, M. J., Adolph, H. W., Zeppezauer, M., and Cedergren-Zeppezauer, E. S. (1996) Eur. J. Biochem. 241, 546-551[Abstract] |
30. | Dunn, M. F., Dietrich, H., MacGibbon, A. K. H., Koerber, S. C., and Zeppezauer, M. (1982) Biochemistry 21, 354-363[Medline] [Order article via Infotrieve] |
31. | Sawaya, M. R., and Kraut, J. (1997) Biochemistry 36, 586-603[CrossRef][Medline] [Order article via Infotrieve] |
32. | Deng, Z., Aliverti, A., Zanetti, G., Arakaki, A. K., Ottado, J., Orellano, E. G., Calcaterra, N. B., Ceccarelli, E. A., Carrillo, N., and Karplus, P. A. (1999) Nat. Struct. Biol. 6, 847-853[CrossRef][Medline] [Order article via Infotrieve] |
33. | Cruickshank, D. W. J.,. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 583-601[CrossRef][Medline] [Order article via Infotrieve] |
34. | Bader, R. F. W. (1990) A Quantum Theory , Oxford University Press, Oxford |
35. | Esnouf, R. M. (1997) J. Mol. Graph Model 15, 132-134[CrossRef][Medline] [Order article via Infotrieve] |
36. | Schaftenaar, G., and Noordik, J. H. (2000) J. Comput. Aided Mol. Design 14, 123-134[CrossRef][Medline] [Order article via Infotrieve] |