Coupling of the guanosine glycosidic bond conformation and the ribonucleotide cleavage reaction: Implications for barnase catalysis (original) (raw)
Related papers
European Journal of Biochemistry, 1993
The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 NEH' activates 02'-H, (d) Glu58 carboxylate acts as base and His92 NEH' as acid in a general acid-base catalysis.
Magnitude of electrostatic interactions in inhibitor binding and during catalysis by ribonuclease A
Biochemistry, 1975
It is demonstrated that a model of nucleotide binding to ribonuclease A similar to that proposed by Hammes and coworkers (G. G. Hammes (1968), Adv. Protein Chem. 23, 1) is, at least, approximately applicable for both cyclic nucleotide substrates and mononucleotide inhibitors at p H values 1 6. 5 and as a function of ionic strength. Calorimetric data on various inhibitors show that the binding reaction can be thermodynamically dissected into a contribution arising from van der Waal's interactions of the nucleoside moiety, characterized by a large negative enthalpy change, and a contribution arising from electrostatic inter-R i b o n u c l e a s e A (RNase) is a structurally well defined enzyme (Richards and Wyckoff, 1971) upon which many investigations relating to its mechanism of action have been focused (Usher and Richardson, 1970; Usher et al., 1972; Witzel, 1963). Numerous studies have provided structural (Wyckoff et al., 1970), thermodynamic (Hammes et al., 1965), and kinetic (Herries et al., 1962; Hammes, 1968) information regarding the interaction of charged substrates and inhibitors with the enzyme. These investigations have clearly demonstrated the importance of electrostatic interactions between the anionic phosphate moiety of nucleotide inhibitors and substrates and the two histidine residues at the catalytic site of the enzyme. It has also been suggested (Witzel, 1963) that similar interactions with other positively charged groups of the enzyme, mainly lysine-41, may play a significant role in both thermodynamic stabilization of the complex and in the catalytic reaction. Most chemical mechanisms which have been proposed to describe the catalytic action of RNase include the formation of a dianionic pentacoordinated phosphate intermediate in the reaction scheme. The thermodynamic significance of such an intermediate is that it can potentially be stabilized by the positively charged environment of the catalytic site. In this communication the magnitude of such electrostatic stabilization and its possible importance in the catalytic reaction are estimated. Experimental Section Ribonuclease A (RNase) was purchased from Worthington Biochemical Corporation and used without further purification. Chromatographically pure cytidine, 2'-cytosine monophosphate (2'-CMP), and 3'-cytosine monophosphate
Proteins, 1989
Molecular dynamics simulations were performed on ribonuclease T1 (RNase T1; EC 3.1.27.3) to determine a structure for the free enzyme. Simulations starting with the X-ray coordinates for the 2'GMP-RNase T1 complex were done in vacuo and with an 18-A water ball around the active site using stochastic boundary conditions to understand the influence of water on both the structure and fluctuations of the enzyme. Removal of 2'GMP caused structural changes in the loop regions, including those directly interacting with the bound inhibitor in the crystal structure, while regions of secondary structure were less affected. The presence of solvent in the simulation damped the structural changes observed, which may be related to the use of full charges in both simulations. Fluctuations were also affected by the water, which generally increased both a t the surface and in the interior of the protein. The active site in vacuo collapsed upon itself, forming a number of protein-protein hydrogen bonds leading to larger structural changes and lowered fluctuations while the presence of water kept the active site open, minimized structural changes, and increased fluctuations. Such fluctuations in the active site may be important for the binding of inhibitors 'This work was in by a postdoctoral a dynamically stable structure, as evidenced by a structure fellowship from the NSF to A.D.M. and grants from the Knut and Alice Wallenberg Foundation, the Swedish Natural Science Research Council, fluctuating around a potential energy is and the Lisa and Johan Gronberg Foundation and in Berlin by the Sonderforschungsbereich 9 and by Fonds der Chemischen Industrie.
Dehydration of Ribonucleotides Catalyzed by Ribonucleotide Reductase: The Role of the Enzyme
Biophysical Journal, 2006
This article focuses on the second step of the catalytic mechanism for the reduction of ribonucleotides catalyzed by the enzyme Ribonucleotide Reductase (RNR). This step corresponds to the protonation/elimination of the substrate's C-29 hydroxyl group. Protonation is accomplished by the neighbor Cys-225, leading to the formation of one water molecule. This is a very relevant step since most of the known inhibitors of this enzyme, which are already used in the fight against certain forms of cancer, are 29-substituted substrate analogs. Even though some theoretical studies have been performed in the past, they have modeled the enzyme with minimal gas-phase models, basically represented by a part of the side chain of the relevant amino acids, disconnected from the protein backbone. This procedure resulted in a limited accuracy in the position and/or orientation of the participating residues, which can result in erroneous energetics and even mistakes in the choice of the correct mechanism for this step. To overcome these limitations we have used a very large model, including a whole R1 model with 733 residues plus the substrate and 10 Å thick shell of water molecules, instead of the minimal gas-phase models used in previous works. The ONIOM method was employed to deal with such a large system. This model can efficiently account for the restrained mobility of the reactive residues, as well as the long-range enzyme-substrate interactions. The results gave additional information about this step, which previous small models could not provide, allowing a much clearer evaluation of the role of the enzyme. The interaction energy between the enzyme and the substrate along the reaction coordinate and the substrate steric strain energy have been obtained. The conclusion was that the barrier obtained with the present model was very similar to the one previously determined with minimal gas-phase models. Therefore, the role of the enzyme in this step was concluded to be mainly entropic, rather than energetic, by placing the substrate and the two reactive residues in a position that allows for the highly favorable concerted trimolecular reaction, and to protect the enzyme radical from the solvent.
Biochemistry, 2005
We have used nucleobase substitution and kinetic analysis to test the hypothesis that hammerhead catalysis occurs by a general acid-base mechanism, in which nucleobases are directly involved in deprotonation of the attacking 2′-hydroxyl group and protonation of the 5′-oxygen that serves as the leaving group in the cleavage reaction. We demonstrate that simultaneous substitution of two important nucleobases, G8 and G12, with 2,6-diaminopurine shifts the pH optimum of the cleavage reaction from greater than 9.5 to approximately 6.8 in two different hammerhead constructs. Controls involving substitution with other nucleobases and combinations of nucleobases at G5, G8, and/or G12 do not show this behavior. The observed changes in the pH-rate behavior are consistent with a mechanism in which N1 protonation-deprotonation events of guanine or 2,6-diaminopurine at positions 8 and 12 are essential for catalysis. Further support for the participation of G8 and G12 comes from photochemical crosslinking experiments, which show that G8 and G12 can stack upon the two substrate nucleobases at the reactive linkage, G(or U)1.1 and C17 (
A guanine nucleobase important for catalysis by the VS ribozyme
The EMBO journal, 2007
A guanine (G638) within the substrate loop of the VS ribozyme plays a critical role in the cleavage reaction. Replacement by any other nucleotide results in severe impairment of cleavage, yet folding of the substrate is not perturbed, and the variant substrates bind the ribozyme with similar affinity, acting as competitive inhibitors. Functional group substitution shows that the imino proton on the N1 is critical, suggesting a possible role in general acid-base catalysis, and this in accord with the pH dependence of the reaction rate for the natural and modified substrates. We propose a chemical mechanism for the ribozyme that involves general acid-base catalysis by the combination of the nucleobases of guanine 638 and adenine 756. This is closely similar to the probable mechanism of the hairpin ribozyme, and the active site arrangements for the two ribozymes appear topologically equivalent. This has probably arisen by convergent evolution.
Archives of Biochemistry and Biophysics, 2002
pH-Dependent kinetic studies were performed with ribonuclease T 1 (RNase T 1 ) and its Glu46Ser, Lys41Met, and Lys41Thr mutants with GpC and polyinosinic acid (PolyI) as substrates. Plots of pH versus logðk cat =K M Þ for both substrates had ascending slopes that were significantly greater for RNase T 1 compared with Glu46Ser-RNase T 1 , which indicated that the c-carboxyl group of conserved Glu46 must be deprotonated (anionic) for maximal interaction with N(1)H and N(2)H of the guanine moiety of GpC or the N(1)H of the hypoxanthine moiety of PolyI. The involvement of the e-ammonium group of nonconserved Lys41 at the 2p subsite (i.e., for an RNA phosphate group two nucleotide positions 5 0 -upstream from the active site) was supported by comparisons of Lys41Met-RNase T 1 and Lys41Thr-RNase T 1 with wild-type. These mutants shared identical catalytic properties (i.e., k cat and K M ) with wild-type using GpC as a substrate. However, k cat =K M for both were identical with each other but lower than those for wild-type when PolyI was the substrate (PolyI has a phosphate group that could interact at a putative 2p site). The pH dependence of this latter difference can be interpreted as reflecting the loss of the 2p subsite interaction with the wild-type enzyme upon deprotonation of the e-ammonium group of Lys41. Subsite interactions for ribonucleases are shown to mainly increase k cat and result in an attenuated pH dependence of k cat =K M . Ó
Chemistry & Biology, 2007
Recent studies indicate that RNA function can be enhanced by the incorporation of conformationally restricted nucleotides. Herein, we use 8-bromoguanosine, a nucleotide analog with an enforced syn conformation, to elucidate the catalytic relevance of ribozyme structures. We chose to study the lead-dependent ribozyme (leadzyme) because structural models derived from NMR, crystal, and computational (MC-Sym) studies differ in which of the three active site guanosines (G7, G9, or G24) have a syn glycosidic torsion angle. Kinetic assays were carried out on 8BrG variants at these three guanosine positions. These data indicate that an 8BrG24 leadzyme is hyperactive, while 8BrG7 and 8BrG9 leadzymes have reduced activity. These findings support the computational model of the leadzyme, rather than the NMR and crystal structures, as being the most relevant to phosphodiester bond cleavage.