J. Am. Chem. zyxwvut SOC. 1995,117, zyxwvu 10365-10372 10365 Initial State of an Enzymatic Reaction. Theoretical Prediction of Complex Formation in the Active Site of RNase T1 zyxwvutsrqponmlkjihgfed F. Cordes, E. B. Starikov, and W. Saenger* Contribution from the Institut f i r Kristallographie, Freie Universitat Berlin, Takustrasse 6, 0-14195 Berlin, Germany Received April 24, 1 9 9 9 Abstract: A computer model for the hydrated complex between the enzyme ribonuclease (RNase) T1 and its substrate zy guanylyl-3’,5’-guanosine has been refined using molecular dynamics simulation and quantum chemical calculations. Actual protonation states of the most important residues at the active site in the presence of the substrate were derived from published NMR titrations and pH-dependent kinetic studies, which were confirmed by independent Monte Carlo calculations (manuscript in preparation). The molecular dynamics trajectory has been analyzed to theoretically capture the initial point of the enzymatic reaction pathway. The changes in the charge distribution of the most relevant part of the enzyme-substrate complex have been checked by the CNDO/Zspd technique. The initial point of the enzymatic reaction pathway has been found to correspond, as expected, to the strained conformation of the “substrate zyxwvutsrq + active site side chains” complex. His40, Glu58, Arg77, and His92 which are primarily involved in the enzymatic activity show hydrogen bond contacts to the substrate. In this scheme, Glu58 plays the role of general base and His92 acts as the general acid in the reaction pathway, while the other two residues stabilize the initial state of the reaction electrostatically. Introduction A variety of structural and functional studies have led to a general view on the mechanism of di-, oligo- and polyribo- nucleotide phosphodiester hydrolysis catalyzed by ribonucleases (RNases).’-3 Because enzyme-substrate and enzyme-transi- tion state complexes have very short lifetimes and thus cannot be captured by conventional experimental techniques, computer modeling is widely used to reconstruct the most salient features of the corresponding enzymatic reaction pathway?-’ Although much is known about the actual mechanism of RNase catalytic activity, details still remain a matter of debate.3 First of all, prior to clarifying these details using methods for enzymatic activity a plausible model for the initial state of the enzymatic reaction under study must be worked out in an independent way. Specifically, the P-05’ phosphoester bond hydrolysis cata- lyzed by RNases is known to be governed by a combination of “general base” and “general acid” residues in the active sites of these enzymes, and is recognized to proceed in two * To whom correspondence should be addressed. FAX: +49 30 838 @ Abstract published in Advance ACS Abstracts, October 1, 1995. (1) Fersht, A. Enzyme Structure and Mechanism; W. H. Freeman: New York, 1985; 426-433. (2) Heinemann, U.; Hahn, U. Topics in molecular and structural biology: Protein nucleic acid interactions; Saenger, W.; Heinemann, U., Eds.; 1989; 67 02. Vol. 10, pp 111-141. (3) Saenger, W. Curr. $pin. Struct. Bioi. zyxwvutsrq 1991, I, 130-138. (4) Holmes, R. R.; Dieters, J. A.; Galucci, J. C. J. Am. Chem. SOC. 1978, (5) Balaji, P. V.; Saenger, W.; Rao, V. S. R. J. Biomol. Struct. Dyn. (6) Deakyne, C. A.; Allen, L. C. J. Am. Chem. SOC. 1979, zyxwvutsrq 101, 3951- (7) Haydock, K.; Lim, C.; Briinger, A. T.; Karplus, M. J. Am. Chem. (8) Tapia, 0.; Andres, J.; Safont, V. S. J. Phys. Chem. 1994, 98,4821- (9) Lee, F. S.; Chu, Z. T.; Warshel, A. J. zyxwvutsrqponm Comput. Chem. 1993, 14, 161- (10) Tempszyk, A.; Tamowka, M.; Liwo, A,; Borowski, E. Eur. Biophys. 100, 7393-7402. 1991, 9, 215-231. 3959. Soc. 1990, 112, 3826-3831. 4830. 185. J. 1992, 21, 137-145. 0002-786319511517-10365$09.00/0 stages: (a) transphosphorylation starting from dinucleoside- monophosphate monoanion and resulting in nucleoside-2‘,3‘- cyclophosphate monoanion formation; (b) hydrolysis starting from nucleoside-2’,3’-cyclophosphate and resulting in nucleo- side-3’-monophosphate formation. For the pyrimidine-specific RNase A two histidines (protonated His1 19+ and unprotonated His 12) play the role of general acid and general base according to the conventional mechanism,’ whereas in the guanine-specific RNase T1 the protonated His92+ and the anionic Glu58- residues stand for the conventional choice of the general acid and general base, respectively.2 Studies on the RNase T1-guided reaction kinetics”,’2 have indicated a residual activity upon genetic substitution of Glu58- to a neutral amino acid. The pH-dependent kinetics of these mutantsI2 together with the knowledge of the orientation of His40 with respect to the ribose derived from X-ray structures of enzyme-inhibitor com- ple~es’~-’~ show that in this case the deprotonated His40 may become the general base in the RNase T1 active site, similar to His12 in RNase A. A proposal that His40 is essential for the reaction and may serve as the general base was also supported by the fact that His40Ala and His92Ala mutants show no activity, whereas the G l ~ 5 8 A l a ” ? ’ ~ mutant possesses some residual activity. pH-dependent kinetic studies for wild-type RNase T1’* showed that His40 is required to be protonated for optimal activity, and led to the conclusion that the protonated His40f must be directly involved in the electrostatic orientation of Glu58- and of the negatively charged substrate;2-’2 the actual role of this residue is not totally clarified at present3 Another problem is connected with the preferential direction (11) Nishikawa, S.; Morioka, H.; Kim, H. J.; Fuchimura, K.; Tanaka, T.; Uesugi, S.; Hakoshima, T.; Tomita, K.; Ohtsuka, E.; Ikehara, M. Biochemistry 1987, 26, 8620-8624. (12) Steyaert, J.; Hallenga, K.; Wyns, L.; Stanssens, P. Biochemistry 1990, 29, 9064-9072. (13) Heinemann, U.; Saenger, W. Nature 1982, 299, 27-31. (14) Koepke, J.; Maslowska, M.; Heinemann, U.; Saenger, W. J. Mol. Bioi. 1989, 206, 475-488. (15) Heydenreich, A.; Koellner, G.; Choe, H. W.; Cordes, F.; Kisker, C.; Schindelin, H.; Adamiak, R.; Hahn, U.; Saenger, W. Eur. J. Biochem. 1993, 218, 1005-1012. 0 1995 American Chemical Society