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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Computer Modelling Studies on the Mechanism of Action of Ribonuclease T1 a
b
P. V. Balaji , W. Saenger & V. S.R. Rao
a
a
Molecular Biophysics Unit , Indian Institute of Science , Bangalore , 560 012 , India b
Institute for Crystallography Free University Berlin , Takustrasse 6, D-1000 , Berlin 33 , Germany Published online: 21 May 2012.
To cite this article: P. V. Balaji , W. Saenger & V. S.R. Rao (1991) Computer Modelling Studies on the Mechanism of Action of Ribonuclease T1 , Journal of Biomolecular Structure and Dynamics, 9:2, 215-231, DOI: 10.1080/07391102.1991.10507908 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507908
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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Journal of Biomolecular Structure & Dynamics, /SSN 0739·11 02 Volume 9, Issue Number 2 (1991), '"Adenine Press (1991).
Computer Modelling Studies on the Mechanism of Action of Ribonuclease T 1 P.V. Balaji\ W. Saenge? and V.S.R. Rao 1* Downloaded by [University of Toronto Libraries] at 06:31 13 January 2015
1
Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012, India
2
Institute for Crystallography Free University Berlin Takustrasse 6, D-1000 Berlin 33, Germany
Abstract The mechanism of action of ribonuclease (RN ase) T 1 is still a matter of considerable debate as the results ofx-ray, 2-D nmr and site-directed mutagenesis studies disagree regarding the role of the catalytically important residues. Hence computer modelling studies were carried out by energy minimisation of the complexes ofRN ase T 1 and some ofits mutants (His40Ala, His40Lys, and Glu58Ala) with the substrate guanyl cytosine (GpC), and of native RNase T 1 with the reaction intermediate guanosine 2', 3' -cyclic phosphate (G >p ). The puckeringofthe guanosine ribose moiety in the minimum energy conformer of the RN ase T 1 - GpC (substrate) complex was found to be 04'-endo and not C3'-endo as in the RNase T 1 - 3'-guanylic acid (inhibitor/product) complex. A possible scheme for the mechanism of action ofRN ase T 1 has been proposed on the basis of the arrangement of the catalytically important amino acid residues His40, Glu58, Arg77, and His92 around the guanosine ribose and the phosphate moiety in the RNase T 1 - GpC and RNase T 1 - G >p complexes. In this scheme, Glu58 serves as the general base group and His92 as the general acid group in the transphosphorylation step. His40 may be essential for stabilising the negatively charged phosphate moiety in the enzyme-transition state complex.
Introduction
Ribonuclease (RNase) T 1 (EC 3.1.27.3), an extracellular enzyme secreted by the fungus Aspergillus oryzae, is a small protein consisting of 104 amino acid residues of known sequence (1 ]. It catalyses the hydrolysis of the phosphoester bonds in single stranded RNA in a two step (transphosphorylation and hydrolysis) reaction involving the intermediate formation of guanosine 2', 3' -cyclic phosphate, G >p. The enzyme shows high specificity towards guanine nucleotides i.e., hydrolysis of the P-05' bonds on the 3'-side of only the guanine nucleosides are catalysed by this enzyme [2]. In the transphosphorylation step, the guanosine 2'-0H group is deprotonated by a general base group and the P-05' bond is cleaved by the attack of the nucleophilic
215
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Balaji et a/.
&Ws' ~
G
o-f~
~
a-
vo
0
s'
o+-
s'
G
-
I I 0
o-+
0-P-0
O=P-0-
l
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AH"'../
OH
Figure l: Mechanism of action ofRNase T 1 proposed from x-ray diffraction, 2-D nmr and site-directed mutagenesis studies. General Base B-
General Acid AH+
Technique
Reference
Glu58
His92
[3] [11]
His40
His92
Glu58
His40
X-ray Site directed mutagenesis Site directed mutagenesis 2-D nmr
[10] [9]
2' -oxygen atom leading to the formation of G >p intermediate. In the hydrolysis step the cyclic phosphate intermediate is hydrolysed to terminal3'-phosphate by the attack of a water molecule [3]. Guanosine 3'-monophosphate (3'-GMP) and oligonucleotides with terminal3'-GMP are released as products of the reaction. The first step is fast and reversible whereas the second is slow and irreversible. As a consequence the cyclic phosphate intermediate gets accumulated and can be isolated [4]. Chemical modification studies have indicated that the amino acid residues His40, Glu58, Arg77 and His92 are essential for catalysis [5]. Based on the x-ray crystallographic studies of RNase T 1 complexed with the inhibitor guanosine 2'monophosphate (2'-GMP) [6]. a scheme for the mechanism of action of the enzyme was proposed in which Glu58 and His92 were implied to act as the general base and general acid groups respectively [7 ,8] (Figure 1). 2-D nmr studies ofRN ase T 1 and its complexes with 2'-GMP and 3'-GMP led to the proposal of a second scheme in which His40 plays the role of the general acid group instead of His92 [9]. The mechanism schemes proposed on the basis of site-directed mutagenesis studies [10,11] also differ from one another. In one of the schemes, His40 and His92 were assigned the roles of the general base and general acid groups respectively [10] whereas in the other scheme, Glu58 and His92 were proposed to serve as the general base and general acid groups respectively [11,12]. Thus the mechanism of action of RNaseT 1 is still a matter of considerable debate. Because of its small size, it is an ideal system for computer modelling to study its selective recognition of guanosine nucleotides and the chemical mechanism of phosphodiester hydrolysis. Hence energy minimisation calculations have been carried out on the complexes ofRN ase
Mechanism of Action of Ribonuclease T1
217
T 1 with dinucleotide substrates and G>p, the cyclic phosphate intermediate. The calculations were also extended to some of the mutants ofRNase T 1• Such studies provide a framework in which the results of x-ray, 2-D nmr and site-directed mutagenesis studies can be integrated to determine the precise role played by the catalytically important residues in the transphosphorylation step and to obtain a better understanding of the RNase T 1 catalysed reaction.
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Methods
The coordinates ofRNase T 1 refined at 1.9 A resolution to a crystallographic Rvalue of0.18 in the RNase T 1-2'-GMP complex [13] served as the initial model for the enzyme. For generating the coordinates for the His40Ala (H40A) and Glu58Ala (E58A) mutants, the side chain atoms of the respective amino acid residues were replaced by a methyl group. The lysine side chain atoms in the His40Lys (H40K) mutant were fixed using standard geometry [14] in staggered orientation. All the CH, CH 2 and CH 3 groups in the protein were treated as united atoms. The coordinates for the explicitly considered polar hydrogen atoms and the dinucleotide substrates (Figure 2) were generated using standard bond lengths and bond angles [15-17]. The torsion angle values used to generate the ribose coordinates were taken from Arnott and Hukins [18]. X-ray crystal structure and CND0/2 optimised [19-21] data were used for generating the coordinates for the ribose-2', 3'-cyclic phosphate moiety. Both the C2' -en do and C3' -en do puckered forms were considered for the two ribose
os-'-H
03'
I
05-H
02
tf
1
03
1
~/
1~\ 02
01-F?-02
if. 05
. 01
··~
Guanosin. i, 3 cyclic phosphat• (G>pl 1
03'
l
r .
Guani,. dinucl•otid• (GpN) Figure 2: Schematic diagram ofGpN and G>p.
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moieties of the substrate dinucleotides and all the different ribose puckered forms of G >p observed in solid state were considered forG >p. In the x-ray diffraction study of the RNase T 1 - 2'-GMP complex [6], most of the water molecules were found around the surface of the protein and sparsely in the active site, and the inhibitor binding site was found to be part of an apparentlyunderhydrated surface portion. Hence instead of including the solvent molecules explicitly, the effect of solvent in damping the electrostatic forces was modelled by using a distance dependent dielectric constant which weighs the short range interactions more than the long range interactions. The modelling studies were carried out in three steps: (i) Contact criteria, (ii) Energy minimisation in the torsion angle space, and (iii) Energy minimisation in the Cartesian coordinate space. Contact criteria and energy minimisation in the torsion angle space were carried out as described earlier [22]. Low energy conformers obtained by energy minimisation in the torsion angle space were taken as starting points for energy minimisation in the Cartesian coordinate space. The total conformational energy which includes the intramolecular interaction energy of the protein and the substrate/intermediate (comprising bond stretching, bond angle bending, van der Waals, electrostatic, hydrogen bonding, and normal and improper torsion potential terms) and intermolecular interaction energy between the enzyme and the substrate/ intermediate was calculated using the expression
The electrostatic (Eete), hydrogen bond (Ehb), and van der Waals (Evw) contributions were evaluated using the functions and parameters described earlier [22]. The partial atomic charges for the amino acid residues are taken from Re( 22 and for the substrate and intermediate were calculated by the CND0/2 method [23,24] (Tables 1,11). A bond torsional model was used for calculating the torsional potential, Etor Imsproper torsion potential (Eimptor) was used to maintain planarity and to avoid racemisation Table I Partial Atomic Charges for the Nucleotide Bases ATOM
ADENINE
ATOM
CYTOSINE
ATOM
GUANINE
ATOM
URACIL
Nl C2 N3 C4 C5 C6 N7 C8 N9 C2-H N6 N6-H C8-H
-.2776 .2217 -.2531 .2088 -.0603 .2656 -.2060 .1638 -.1242 -.0403 -.2327 .1207 -.0126
Nl C2 N3 C4 C5 C6 02 N4 N4-H C5-H C6-H
-.1562 .4215 -.3403 .3210 -.1653 .1918 -.4132 -.2274 .1261 .0165 -.0061
Nl C2 N3 C4 C5 C6 N7 C8 N9 NI-H N2 N2-H 06 C8-H
-.2139 .3815 -.3272 .2117 -.1020 .3529 -.1652 .1342 -.1202 .1205 -.2474 .1357 -.3890 -.0128
Nl C2 N3 C4 C5 C6 02 N3-H 04 C5-H C6-H
-.1720 .4515 -.2382 .3704 -.1571 .1762 -.3673 .1402 -.3507 .0381 .0034
219
Mechanism of Action of Ribonuclease T1
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Table II Partial Atomic Charges for the Ribose-Phosphate Moieties ATOM
(l)'
(2)'
(3)'
Cl' HI' C2' H2' C3' H3' C4' H4' 04' 02' 02'-H 03' C5' H5' 05' 05'-H p P-Ol P-02
.2377 -.0539 .1127 -.0260 .1546 -.0238 .1260 .0127 -.2681 -.2360 .1071 -.2821 .1238 .0111 -.2560 .1074 .3792 -.4424 -.4424
.2373 -.0448 .1205 -.0352 .1362 -.0240 .1176 -.0042 -.2403 -.2508 .1254 -.2508 .1535 -.0134 -.2807
.2224 -.0220 .1423 -.0405 .1268 -.0333 .1249 -.0031 -.2598 -.2826
.3792 -.4424 -.4424
-.2771 .1453 -.0311 -.2437 .1122 .3665 -.4553 -.4553
1(1)
Phosphodiesterbond on 3'-oxygen; unsubstituted 5'-0H group. (2) Phosphodiester bond on 5' -oxygen; unsubstituted 3'-OH group. (3) Ribose-phosphate moiety in guanosine-2', 3'-cyclic phosphate.
at chiral centers. Bond lengths and bond angles were restrained to their equilibrium values by harmonic potential functions (~ 1 and ~ 3 ). The form of the function and the parameters used are the same as that given by Weiner et al. [25,25]. A residue based non bonded cutoff of 10 Awas used for evaluating the intramolecular protein energy. For each residue, the list of other residues within the cutoff distance was updated at the end of every 200 iterations of minimisation. A modification of the conjugate gradient algorithm as suggested by Shanno [27] was used for minimisation and the gradients were calculated by analytical differential of the energy function. Minimisation was terminated when the rms gradient is less than 0.01 kcal/mol A.
Results and Discussion The results of the energy minimisation of the RNase T 1 complexes with the four dinucleotides GpA, GpC, GpG and GpU are shown in Tables III and N. These tables show that these substrate molecules assume very similar orientations in the binding site of the enzyme and form the same hydrogen bonds with the protein especially around the guanosine ribose and the phosphate groups irrespective of the nature of the second base. This corroborates the steady state kinetic studies of the RNase T 1 catalysed transesterification ofGpA, GpC, GpG and GpU [28,29] which indicated that the values ofkcalkm for the four GpNs are virtually identical. In viewofthis, energy minimisation in the Cartesian coordinate space was restricted to only the complexes of GpC with native and some mutants ofRNase T 1 (H40A, H40K and E58A).
220
Balaji et a/. Table III* Orientation and Conformational Angles of Substrate GpNs in the Minimum Energy Conformers ofRNase T 1 - GpN Complexes
Substrate Dinucleotide
GpA
GpC
GpG
GpU
Translational Parameters X
y
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z Rotational Parameters (in degrees) p. It is interesting to note that in RNase A, Lys41 which is important for stabilising the transition state is found to be near 02' of the cyclic phosphate intermediate [47,48]. It thus seems that it is the positive charge of His40 which is essential for the enzyme activity perhaps for stabilising the RNase T 1 - transition state complex. Such a role for His40 is also consistent with the observed I 0% activity of the H40K-RNaseT 1 mutant and the inactivity of the H40A- and H40D-RNaseT 1 mutants [10-12].
Proposed Scheme for Mechanism ofAction of RNase T1 A probable scheme for the mechanism of action ofRNase T 1 can be postulated on the basis of the relative positions ofHis40, Glu58 and His92 with respect to GpC and G>p in their respective complexes with RNase T 1 (Figure 10).
K
tl
E58
ESI
·-·-=ooc ~ H........-ooc ~ H92 lm-H-·--·0- P-o ......--- lm .. _...HQ- -o .~ H92
e _
I
J
R7,.
H4Cf
·x;:r·
R77•j
N
\'Y nH
H40
0
1"1'
OH
l'f' TRANSPHOSPHORYLAT~
~oyG
H
H92
•
lm-H .....__ _ _ _
OHN I 'o-
R~
0
~
~6
ESI
0 0 lOX 0 -~p"H4o•
H
0
H92 lm
0 H40•
' { ESI
-l '\oHOOC
R77•
··~ 0
+-
CJi
Figure 10: Scheme for the mechanism of action ofRNase T 1 proposed from the present study.
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Balaji et a/.
When a substrate molecule like GpC binds to RNase T 1, the guanosine ribose moiety will be distorted to an 04' -en do pucker from its initial C2' -endo or C3 '-en do puckered form. Glu58 in its ionised form, will be positioned close to the guanosine 2' -OH group and the protonated His92 residue will be close to one of the phosphate oxygen atoms. His92 protonates the phosphate group and Glu58 deprotonates the 2' -OH group simultaneously. The protonated His40 residue will probably stabilise the penta-coordinated transition state. The proton from the phosphate is removed by His92 and transferred to the 05' of the leaving group nucleoside i.e., cytidine moietyofGpC leading to the formation of the cyclic phosphate intermediate. In the enzyme-intermediate complex, the protonated Glu58 hydrogen bonds with the phosphate group and His40 is near the 2' -oxygen atom. The hydrolysis step is initiated by the attack of the phosphate group by a water molecule and essentially follows the reverse sequence of the first step. Arg77 forms hydrogen bonds with the phosphate group in all the three i.e., enzyme-substrate, enzyme-intermediate and enzyme-product complexes. A very similar scheme has been proposed by Breslow and coworkers from kinetic studies on model systems for the mechanism of action of RNase A catalysed reaction (32-34].
Conclusions The present calculations show that most probably the amino acid residues Glu58 and His92 serve as the general base and acid groups for the RNase T 1 catalysed hydrolysis reaction and His40 perhaps is essential for stabilising the transition state, and this is consistent with the conclusions of chemical modification, physicochemical and x-ray diffraction studies. The scheme for the mechanism of action ofRN ase T 1 proposed here differs in detail from the earlier ones.
Acknowledgements The authors thank Prof. G. Govil, Tata Institute of Fundamental Research (TIFR), Bombay, for very generously allowing them to use the Silicon Graphics Workstation IRIS-4D of the 500 MHz FT NMR Facility funded by the Department of Science and Technology and located at TIFR, Bombay. W.S. gratefully acknowledges support by the DFG (Schwerpunkt program "Protein-Design"). References and Footnotes
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
K. Takahashi,] Biochem. 98, 815 (1985). K. Sato-Asano,J Biochem. 46,31 (1959). U. Heinemann and W. Saenger, Pure and App. Chern. 57, 417 ( 1985). K. Takahashi and S. Moore, The Enzymes XV, 435 (1982). F. Egami, T. Oshima and T. Uchida, Mol. Bioi. Biochem. Biophys. 32, 250 (1980). R. Ami, U. Heinemann, R. Tokuoka and W. Saenger,] Bioi. Chern. 263, 15358 (1988). U. Heinemann and W. Saenger, Nature 299,27 (1982). U. Heinemann and W. Saenger,] Bioi. Str. Dyn. 1, 523 (1983). E. Hoffmann, J. Schmidt, J. Simon and H. Riiterjans, Nucleosides and Nucleotides 7, 757 (1988). S. Nishikawa, H. Morioka, H. Kim, K. Fuchimura, T. Tanaka, S. Uesugi,T. Hakoshima, K. Tomita, E. Ohtsuka and M. Ikehara, Biochem. 26, 8620 (1987). 11. J. Steyaert, C. Thoeu and P. Stanssens, In Structure and Chemistry ofRibonucleases, Proceedings of
the first international meeting, Moscow 216 (1989).
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Mechanism of Action of Ribonuclease T1 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.
45. 46. 47. 48.
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J. Steyaert, K. Hallenga, L. Wyns and P. Stanssens, Biochem. 29,9064 (1990). RAmi, U. Heinemann, M. Maslowska, R. Tokuoka and W. Saenger, Acta Cryst. B43, 548 (1987). F.A. Momany, R.F. McGuire, AW. Burgess and H.A. Scheraga,J Phys. Chem. 79, 2361 (1975). E.N. Baker and R.E. Hubbard, Prog. Biophys. Mol. Bioi. 44, 97 (1984). R. Taylor and 0. Kennard,.!. Mol. Str. 78, 1 (1982). W. Saenger, Principles of Nucleic Acid Structure, Springer Verlag, first edition, Berlin (1982). S. Arnott and D.W.L. Hukins, Biochem. J. 130, 453 (1972). C.L. Coulter,.!. Am. Chem. Soc. 95,570 (1973). B.S. Reddy and W. Saenger, Acta Cryst. B34, 1520 (1978). H. Umeyama, S. Nakagawa and T. Fujii, Chem. Pharm. Bull. 27,974 (1979). P.V. Balaji, W. Saenger and V.S.R. Rao, Biopoly. 30, 257 (1990). J.A Pople and D.L. Beveridge, Approximate Molecular Orbital Theory, McGraw Hill, New York (1970). W.J. Hehre, W.A. Lathan, R. Ditchfield, M.D. Newton and J.A Pople, Quantum Chemistry Program Exchange, No. 236 (1975). S.J. Weiner, P.A. Kollman, D.A. Case, U.C. Singh, C. Ghio, G. Alagona, S. Profeta, Jr. and PJ. Weiner,.!. Am. Chem. Soc. 106, 765 (1984). SJ. Weiner, P.A. Kollman, D.T. Nguyen and D.A. Case,.!. Comp. Chem. 7, 230 (1986). D.F. Shanno, Math. Opr. Res. 3, 244 (1978). M. Zabinski and F.G. Walz, Jr., Arch. Biochem. Biophys. 175, 558 (1976). H.L. Osterman and F.G. Walz, Jr., Biochem. 17,4124 (1978). P.V. Balaji, W. Saenger and V.S.R. Rao, Cu". Sci. 60, 363 (1991). F. Inagaki, Y. Kawano, I. Shimada, K. Takahashi and T. Miyazawa,J. Biochem. 89, 1185 (1981). R. Breslow and M. Labelle,.!. Am. Chem. Soc. 108,2655 (1986). E. Anslyn and R. Breslow, J. Am. Chem. Soc. 111. 4473 ( 1989). R. Breslow, D.-L. Huang and E. Anslyn, Proc. Nat/. Acad. Sci. (USA) 86, 1746 (1989). K. Sato and F. Eqami,J. Biochem. 44,753 (1957). F.G. Walz, Jr., Biochem. 16,4568 (1977). S. Nishikawa, H. Morioka, K. Fuchimura, T. Tanaka, S. Uesugi, E.Ohtsuka and M. Ikehara, Biochem. Biophys. Res. Comm. 138, 789 (1986). B.A. Shirley, P. Stanssens, J. Steyaert and C.N. Pace, J. Bioi. Chem. 264, 11621 (1989). M. McNutt, L.S. Mullins, F.M. Raushel and C.N. Pace, Biochem. 29, 7572 (1990). P. Blackburn and S. Moore, The Enzymes XV, 317 (1982). B. Gutte,J. Bioi. Chem. 250,889 (1975). E. Machuga and M.H. Klapper, J. Bioi. Chem. 250,2319 (1975). E. Machuga and M.H. Klapper, Biochim. Biophys. Acta 481, 526 ( 1977). W. Saenger, R. Ami, M. Maslowska, A Paehler and U. Heinemann, In Crystallography in Molecular Biology, D. Moras, J. Drenth, B. Strandberg, D. Suck and K. Wilson, eds., NATO ASI series, Series A: Life Sciences, Vol. 126, Plenum press, New York 337 ( 1985). C. Hill, In Structure and Chemistry ofRibonucleases, Proceedings of the first international meeting, Moscow 171 (1989). C. Hill, G. Dodson, U. Heinemann, W. Saenger, Y. Mitsui, K. Nakamura, S. Borisov, G. Tischenko, K. Polyakov and S. Pavlovsky, Trends in Biochem. Sci. 8, 364 (1983). C. Brooks III, A Bruenger, M. Francl, K. Haydock. L.C. Allen and M. Karplus,Ann. NY. Acad. Sci. 471' 295 (1986). K. Haydock. C. Lim, AT. Bruenger and M. Karplus, J. Am. Chem. Soc. 112, 3826 ( 1990).
Date Received: June 15, 1991
Communicated by the Editor Dino Moras
Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Computer Modelling Studies on the Mechanism of Action of Ribonuclease T1 a
b
P. V. Balaji , W. Saenger & V. S.R. Rao
a
a
Molecular Biophysics Unit , Indian Institute of Science , Bangalore , 560 012 , India b
Institute for Crystallography Free University Berlin , Takustrasse 6, D-1000 , Berlin 33 , Germany Published online: 21 May 2012.
To cite this article: P. V. Balaji , W. Saenger & V. S.R. Rao (1991) Computer Modelling Studies on the Mechanism of Action of Ribonuclease T1 , Journal of Biomolecular Structure and Dynamics, 9:2, 215-231, DOI: 10.1080/07391102.1991.10507908 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507908
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [University of Toronto Libraries] at 06:31 13 January 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Journal of Biomolecular Structure & Dynamics, /SSN 0739·11 02 Volume 9, Issue Number 2 (1991), '"Adenine Press (1991).
Computer Modelling Studies on the Mechanism of Action of Ribonuclease T 1 P.V. Balaji\ W. Saenge? and V.S.R. Rao 1* Downloaded by [University of Toronto Libraries] at 06:31 13 January 2015
1
Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012, India
2
Institute for Crystallography Free University Berlin Takustrasse 6, D-1000 Berlin 33, Germany
Abstract The mechanism of action of ribonuclease (RN ase) T 1 is still a matter of considerable debate as the results ofx-ray, 2-D nmr and site-directed mutagenesis studies disagree regarding the role of the catalytically important residues. Hence computer modelling studies were carried out by energy minimisation of the complexes ofRN ase T 1 and some ofits mutants (His40Ala, His40Lys, and Glu58Ala) with the substrate guanyl cytosine (GpC), and of native RNase T 1 with the reaction intermediate guanosine 2', 3' -cyclic phosphate (G >p ). The puckeringofthe guanosine ribose moiety in the minimum energy conformer of the RN ase T 1 - GpC (substrate) complex was found to be 04'-endo and not C3'-endo as in the RNase T 1 - 3'-guanylic acid (inhibitor/product) complex. A possible scheme for the mechanism of action ofRN ase T 1 has been proposed on the basis of the arrangement of the catalytically important amino acid residues His40, Glu58, Arg77, and His92 around the guanosine ribose and the phosphate moiety in the RNase T 1 - GpC and RNase T 1 - G >p complexes. In this scheme, Glu58 serves as the general base group and His92 as the general acid group in the transphosphorylation step. His40 may be essential for stabilising the negatively charged phosphate moiety in the enzyme-transition state complex.
Introduction
Ribonuclease (RNase) T 1 (EC 3.1.27.3), an extracellular enzyme secreted by the fungus Aspergillus oryzae, is a small protein consisting of 104 amino acid residues of known sequence (1 ]. It catalyses the hydrolysis of the phosphoester bonds in single stranded RNA in a two step (transphosphorylation and hydrolysis) reaction involving the intermediate formation of guanosine 2', 3' -cyclic phosphate, G >p. The enzyme shows high specificity towards guanine nucleotides i.e., hydrolysis of the P-05' bonds on the 3'-side of only the guanine nucleosides are catalysed by this enzyme [2]. In the transphosphorylation step, the guanosine 2'-0H group is deprotonated by a general base group and the P-05' bond is cleaved by the attack of the nucleophilic
215
216
Balaji et a/.
&Ws' ~
G
o-f~
~
a-
vo
0
s'
o+-
s'
G
-
I I 0
o-+
0-P-0
O=P-0-
l
Downloaded by [University of Toronto Libraries] at 06:31 13 January 2015
AH"'../
OH
Figure l: Mechanism of action ofRNase T 1 proposed from x-ray diffraction, 2-D nmr and site-directed mutagenesis studies. General Base B-
General Acid AH+
Technique
Reference
Glu58
His92
[3] [11]
His40
His92
Glu58
His40
X-ray Site directed mutagenesis Site directed mutagenesis 2-D nmr
[10] [9]
2' -oxygen atom leading to the formation of G >p intermediate. In the hydrolysis step the cyclic phosphate intermediate is hydrolysed to terminal3'-phosphate by the attack of a water molecule [3]. Guanosine 3'-monophosphate (3'-GMP) and oligonucleotides with terminal3'-GMP are released as products of the reaction. The first step is fast and reversible whereas the second is slow and irreversible. As a consequence the cyclic phosphate intermediate gets accumulated and can be isolated [4]. Chemical modification studies have indicated that the amino acid residues His40, Glu58, Arg77 and His92 are essential for catalysis [5]. Based on the x-ray crystallographic studies of RNase T 1 complexed with the inhibitor guanosine 2'monophosphate (2'-GMP) [6]. a scheme for the mechanism of action of the enzyme was proposed in which Glu58 and His92 were implied to act as the general base and general acid groups respectively [7 ,8] (Figure 1). 2-D nmr studies ofRN ase T 1 and its complexes with 2'-GMP and 3'-GMP led to the proposal of a second scheme in which His40 plays the role of the general acid group instead of His92 [9]. The mechanism schemes proposed on the basis of site-directed mutagenesis studies [10,11] also differ from one another. In one of the schemes, His40 and His92 were assigned the roles of the general base and general acid groups respectively [10] whereas in the other scheme, Glu58 and His92 were proposed to serve as the general base and general acid groups respectively [11,12]. Thus the mechanism of action of RNaseT 1 is still a matter of considerable debate. Because of its small size, it is an ideal system for computer modelling to study its selective recognition of guanosine nucleotides and the chemical mechanism of phosphodiester hydrolysis. Hence energy minimisation calculations have been carried out on the complexes ofRN ase
Mechanism of Action of Ribonuclease T1
217
T 1 with dinucleotide substrates and G>p, the cyclic phosphate intermediate. The calculations were also extended to some of the mutants ofRNase T 1• Such studies provide a framework in which the results of x-ray, 2-D nmr and site-directed mutagenesis studies can be integrated to determine the precise role played by the catalytically important residues in the transphosphorylation step and to obtain a better understanding of the RNase T 1 catalysed reaction.
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Methods
The coordinates ofRNase T 1 refined at 1.9 A resolution to a crystallographic Rvalue of0.18 in the RNase T 1-2'-GMP complex [13] served as the initial model for the enzyme. For generating the coordinates for the His40Ala (H40A) and Glu58Ala (E58A) mutants, the side chain atoms of the respective amino acid residues were replaced by a methyl group. The lysine side chain atoms in the His40Lys (H40K) mutant were fixed using standard geometry [14] in staggered orientation. All the CH, CH 2 and CH 3 groups in the protein were treated as united atoms. The coordinates for the explicitly considered polar hydrogen atoms and the dinucleotide substrates (Figure 2) were generated using standard bond lengths and bond angles [15-17]. The torsion angle values used to generate the ribose coordinates were taken from Arnott and Hukins [18]. X-ray crystal structure and CND0/2 optimised [19-21] data were used for generating the coordinates for the ribose-2', 3'-cyclic phosphate moiety. Both the C2' -en do and C3' -en do puckered forms were considered for the two ribose
os-'-H
03'
I
05-H
02
tf
1
03
1
~/
1~\ 02
01-F?-02
if. 05
. 01
··~
Guanosin. i, 3 cyclic phosphat• (G>pl 1
03'
l
r .
Guani,. dinucl•otid• (GpN) Figure 2: Schematic diagram ofGpN and G>p.
218
Balaji et a/.
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moieties of the substrate dinucleotides and all the different ribose puckered forms of G >p observed in solid state were considered forG >p. In the x-ray diffraction study of the RNase T 1 - 2'-GMP complex [6], most of the water molecules were found around the surface of the protein and sparsely in the active site, and the inhibitor binding site was found to be part of an apparentlyunderhydrated surface portion. Hence instead of including the solvent molecules explicitly, the effect of solvent in damping the electrostatic forces was modelled by using a distance dependent dielectric constant which weighs the short range interactions more than the long range interactions. The modelling studies were carried out in three steps: (i) Contact criteria, (ii) Energy minimisation in the torsion angle space, and (iii) Energy minimisation in the Cartesian coordinate space. Contact criteria and energy minimisation in the torsion angle space were carried out as described earlier [22]. Low energy conformers obtained by energy minimisation in the torsion angle space were taken as starting points for energy minimisation in the Cartesian coordinate space. The total conformational energy which includes the intramolecular interaction energy of the protein and the substrate/intermediate (comprising bond stretching, bond angle bending, van der Waals, electrostatic, hydrogen bonding, and normal and improper torsion potential terms) and intermolecular interaction energy between the enzyme and the substrate/ intermediate was calculated using the expression
The electrostatic (Eete), hydrogen bond (Ehb), and van der Waals (Evw) contributions were evaluated using the functions and parameters described earlier [22]. The partial atomic charges for the amino acid residues are taken from Re( 22 and for the substrate and intermediate were calculated by the CND0/2 method [23,24] (Tables 1,11). A bond torsional model was used for calculating the torsional potential, Etor Imsproper torsion potential (Eimptor) was used to maintain planarity and to avoid racemisation Table I Partial Atomic Charges for the Nucleotide Bases ATOM
ADENINE
ATOM
CYTOSINE
ATOM
GUANINE
ATOM
URACIL
Nl C2 N3 C4 C5 C6 N7 C8 N9 C2-H N6 N6-H C8-H
-.2776 .2217 -.2531 .2088 -.0603 .2656 -.2060 .1638 -.1242 -.0403 -.2327 .1207 -.0126
Nl C2 N3 C4 C5 C6 02 N4 N4-H C5-H C6-H
-.1562 .4215 -.3403 .3210 -.1653 .1918 -.4132 -.2274 .1261 .0165 -.0061
Nl C2 N3 C4 C5 C6 N7 C8 N9 NI-H N2 N2-H 06 C8-H
-.2139 .3815 -.3272 .2117 -.1020 .3529 -.1652 .1342 -.1202 .1205 -.2474 .1357 -.3890 -.0128
Nl C2 N3 C4 C5 C6 02 N3-H 04 C5-H C6-H
-.1720 .4515 -.2382 .3704 -.1571 .1762 -.3673 .1402 -.3507 .0381 .0034
219
Mechanism of Action of Ribonuclease T1
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Table II Partial Atomic Charges for the Ribose-Phosphate Moieties ATOM
(l)'
(2)'
(3)'
Cl' HI' C2' H2' C3' H3' C4' H4' 04' 02' 02'-H 03' C5' H5' 05' 05'-H p P-Ol P-02
.2377 -.0539 .1127 -.0260 .1546 -.0238 .1260 .0127 -.2681 -.2360 .1071 -.2821 .1238 .0111 -.2560 .1074 .3792 -.4424 -.4424
.2373 -.0448 .1205 -.0352 .1362 -.0240 .1176 -.0042 -.2403 -.2508 .1254 -.2508 .1535 -.0134 -.2807
.2224 -.0220 .1423 -.0405 .1268 -.0333 .1249 -.0031 -.2598 -.2826
.3792 -.4424 -.4424
-.2771 .1453 -.0311 -.2437 .1122 .3665 -.4553 -.4553
1(1)
Phosphodiesterbond on 3'-oxygen; unsubstituted 5'-0H group. (2) Phosphodiester bond on 5' -oxygen; unsubstituted 3'-OH group. (3) Ribose-phosphate moiety in guanosine-2', 3'-cyclic phosphate.
at chiral centers. Bond lengths and bond angles were restrained to their equilibrium values by harmonic potential functions (~ 1 and ~ 3 ). The form of the function and the parameters used are the same as that given by Weiner et al. [25,25]. A residue based non bonded cutoff of 10 Awas used for evaluating the intramolecular protein energy. For each residue, the list of other residues within the cutoff distance was updated at the end of every 200 iterations of minimisation. A modification of the conjugate gradient algorithm as suggested by Shanno [27] was used for minimisation and the gradients were calculated by analytical differential of the energy function. Minimisation was terminated when the rms gradient is less than 0.01 kcal/mol A.
Results and Discussion The results of the energy minimisation of the RNase T 1 complexes with the four dinucleotides GpA, GpC, GpG and GpU are shown in Tables III and N. These tables show that these substrate molecules assume very similar orientations in the binding site of the enzyme and form the same hydrogen bonds with the protein especially around the guanosine ribose and the phosphate groups irrespective of the nature of the second base. This corroborates the steady state kinetic studies of the RNase T 1 catalysed transesterification ofGpA, GpC, GpG and GpU [28,29] which indicated that the values ofkcalkm for the four GpNs are virtually identical. In viewofthis, energy minimisation in the Cartesian coordinate space was restricted to only the complexes of GpC with native and some mutants ofRNase T 1 (H40A, H40K and E58A).
220
Balaji et a/. Table III* Orientation and Conformational Angles of Substrate GpNs in the Minimum Energy Conformers ofRNase T 1 - GpN Complexes
Substrate Dinucleotide
GpA
GpC
GpG
GpU
Translational Parameters X
y
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z Rotational Parameters (in degrees) p. It is interesting to note that in RNase A, Lys41 which is important for stabilising the transition state is found to be near 02' of the cyclic phosphate intermediate [47,48]. It thus seems that it is the positive charge of His40 which is essential for the enzyme activity perhaps for stabilising the RNase T 1 - transition state complex. Such a role for His40 is also consistent with the observed I 0% activity of the H40K-RNaseT 1 mutant and the inactivity of the H40A- and H40D-RNaseT 1 mutants [10-12].
Proposed Scheme for Mechanism ofAction of RNase T1 A probable scheme for the mechanism of action ofRNase T 1 can be postulated on the basis of the relative positions ofHis40, Glu58 and His92 with respect to GpC and G>p in their respective complexes with RNase T 1 (Figure 10).
K
tl
E58
ESI
·-·-=ooc ~ H........-ooc ~ H92 lm-H-·--·0- P-o ......--- lm .. _...HQ- -o .~ H92
e _
I
J
R7,.
H4Cf
·x;:r·
R77•j
N
\'Y nH
H40
0
1"1'
OH
l'f' TRANSPHOSPHORYLAT~
~oyG
H
H92
•
lm-H .....__ _ _ _
OHN I 'o-
R~
0
~
~6
ESI
0 0 lOX 0 -~p"H4o•
H
0
H92 lm
0 H40•
' { ESI
-l '\oHOOC
R77•
··~ 0
+-
CJi
Figure 10: Scheme for the mechanism of action ofRNase T 1 proposed from the present study.
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Balaji et a/.
When a substrate molecule like GpC binds to RNase T 1, the guanosine ribose moiety will be distorted to an 04' -en do pucker from its initial C2' -endo or C3 '-en do puckered form. Glu58 in its ionised form, will be positioned close to the guanosine 2' -OH group and the protonated His92 residue will be close to one of the phosphate oxygen atoms. His92 protonates the phosphate group and Glu58 deprotonates the 2' -OH group simultaneously. The protonated His40 residue will probably stabilise the penta-coordinated transition state. The proton from the phosphate is removed by His92 and transferred to the 05' of the leaving group nucleoside i.e., cytidine moietyofGpC leading to the formation of the cyclic phosphate intermediate. In the enzyme-intermediate complex, the protonated Glu58 hydrogen bonds with the phosphate group and His40 is near the 2' -oxygen atom. The hydrolysis step is initiated by the attack of the phosphate group by a water molecule and essentially follows the reverse sequence of the first step. Arg77 forms hydrogen bonds with the phosphate group in all the three i.e., enzyme-substrate, enzyme-intermediate and enzyme-product complexes. A very similar scheme has been proposed by Breslow and coworkers from kinetic studies on model systems for the mechanism of action of RNase A catalysed reaction (32-34].
Conclusions The present calculations show that most probably the amino acid residues Glu58 and His92 serve as the general base and acid groups for the RNase T 1 catalysed hydrolysis reaction and His40 perhaps is essential for stabilising the transition state, and this is consistent with the conclusions of chemical modification, physicochemical and x-ray diffraction studies. The scheme for the mechanism of action ofRN ase T 1 proposed here differs in detail from the earlier ones.
Acknowledgements The authors thank Prof. G. Govil, Tata Institute of Fundamental Research (TIFR), Bombay, for very generously allowing them to use the Silicon Graphics Workstation IRIS-4D of the 500 MHz FT NMR Facility funded by the Department of Science and Technology and located at TIFR, Bombay. W.S. gratefully acknowledges support by the DFG (Schwerpunkt program "Protein-Design"). References and Footnotes
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
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Mechanism of Action of Ribonuclease T1 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.
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Date Received: June 15, 1991
Communicated by the Editor Dino Moras
