Background: Low binding affinity of product UMP is used to argue against substrate distortion contributing to orotidine-5Ј-monophosphate decarboxylase catalysis. Results: Atomic resolution structure and surface plasmon resonance analysis reveal strong repulsion between active site residue and UMP. Conclusion: Low UMP affinity does not disprove contribution of substrate distortion to catalysis. Significance: Substrate distortion can still be considered as a mechanistic feature of this most proficient enzyme.Orotidine 5-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of its substrate by 17 orders of magnitude. One argument brought forward against steric/electrostatic repulsion causing substrate distortion at the carboxylate substituent as part of the catalysis has been the weak binding affinity of the decarboxylated product (UMP). The crystal structure of the UMP complex of ODCase at atomic resolution (1.03 Å) shows steric competition between the product UMP and the side chain of a catalytic lysine residue. Surface plasmon resonance analysis indicates that UMP binds 5 orders of magnitude more tightly to a mutant in which the interfering side chain has been removed than to wild-type ODCase. These results explain the low affinity of UMP and counter a seemingly very strong argument against a contribution of substrate distortion to the catalytic reaction mechanism of ODCase.Orotidine 5Ј-monophosphate decarboxylase (ODCase) 3 is one of the most proficient enzymes known (1). It decarboxylates orotidine 5Ј-monophosphate (OMP) and produces uridine 5Ј-monophosphate (UMP) in the final step of the de novo pyrimidine biosynthesis pathway (Fig. 1A). It also accelerates the reaction by 17 orders of magnitude, as compared with the spontaneous reaction in water at neutral pH, without employing cofactors or metal ions (1-5).The reaction mechanism of this enzyme has been the subject of extensive investigations. More than 170 crystal structures have been determined, and numerous kinetic assays at various conditions have been performed. These experiments established that an electrostatic residue network composed of the charged side chains of two aspartate and two lysine residues, all completely conserved, plays a dominant role in catalysis (Fig. 1B) (2-5). As with all enzymes, the reaction acceleration provided by ODCase is explained in part by transition state stabilization (6). Lys-72 (the sequence numbers in this study correspond to those of the ODCase from Methanothermobacter thermautotrophicus (MtODCase)) is considered to be the key residue to stabilize the intermediate vinyl anion (6,7). However, there is still no general agreement on all the details of the reaction mechanism. In particular, the observation that ODCase also converts 6-cyano-UMP into 6-hydroxy-UMP at the same site where the decarboxylation reaction occurs (Fig. 1A) (8) complicates the scenario. The environment required to stabilize the vinyl anion does not seem suitable to support, at the same time, the intermediate of the side reaction b...

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One way for enzymes to affect reactions they catalyze is through transition state stabilization. Another factor to be considered is the contribution of substrate distortion, although it has been thoroughly described for only a few enzymes. We have a longstanding interest in the reaction mechanism of orotidine monophosphate decarboxylase (ODCase) and determined various crystal structures bound with distorted substrates at around 1.5 Å resolution. The enzyme is known as one of the most proficient enzymes, which accelerates the decarboxylation of orotidine 5'-monophosphate (OMP) to form uridine 5'-monophosphate (UMP) by 17 orders of magnitude. One argument against the contribution of substrate distortion to the ODCase reaction is the weak affinity of UMP. The distortions observed so far all appear at the C6-substituent of the pyrimidine ring, which corresponds to the carboxylate of OMP. Since the carboxylate is removed by the reaction, the product UMP should bind more tightly to ODCase than OMP, if the distortion of C6-substituent contributes to the catalysis. In order to investigate this inconsistency, we determined the crystal structure of ODCase with UMP at atomic resolution (1.03 Å). The structure showed an unfavorable interaction between UMP and the catalytic residue K72, an interaction considered to be absent in the OMP complex. Surface plasmon resonance analysis indicated that UMP binds stronger to the K72A mutant than to the wild-type enzyme by 5 orders of magnitude. These analyses invalidate the argument against a contribution of substrate distortion to ODCase catalysis. Finally, we estimated how much the distortion contributes to the catalysis using computational simulation methods. The results indicated that 10-15% decrease of the ΔΔG‡ value is contributed by substrate distortion.

The ERK MAP kinase plays a central role in the signaling cascades of cell growth. Here, we show that stochastic ERK activity pulses regulate cell proliferation rates in a cell density-dependent manner. A biosensor based on the principle of fluorescence resonance energy (FRET) revealed stochastic ERK activity pulses fired spontaneously or propagated from adjacent cells. Frequency, but not amplitude, of ERK activity pulses exhibited a bell-shaped response to the cell density and cell proliferation rates. Consistently, synthetic ERK activity pulses generated by a lightswitchable CRaf protein accelerated cell proliferation. Taken together, these findings reveal a role of the stochastic ERK activity pulses in cell proliferation. 1SCA-06 細胞内シグナル伝達経路の情報コーディング Information coding of cellular signaling networksShinya Kuroda, Shinsuke Uda (Biophys. Biochem., University of Tokyo) Cellular signaling network can be regarded as a communication channel in the framework of Shannon's information theory. We can measure the distribution of phosphorylation of ERK and CREB and expression of IEGs products at a cell population level. We found that information transmission was generally more robust than averaged signal intensity despite pharmacological perturbations, and information transmission through unperturbed signaling pathways compensatorily increased in many signaling pathways. We propose that cells use information entropy as information, so that messages can be robustly transmitted despite noise and variation in molecular activities between individual cells. Information coding will be discussed as a general property of cellular signaling. In bacteria, SecA, the translocation ATPase and SecYEG, the polypeptideconducting channel, play central roles in protein translocation across the cytoplasmic membrane. Membrane proteins SecDF, conserved throughout the bacterial kingdom, form a complex with SecYEG and are required for efficient protein export. Thus, it is important to elucidate mechanisms of the SecDF enhancement of translocation. We discuss structure and function of SecDF on the basis of the crystal structure of the T. thermophilus ortholog and structure-instructed biochemical analyses of the E. coli system including site-directed in vivo photo-crosslinking. Based on the results, we propose that SecDF functions as a cation-driven molecular motor to pull a translocating polypeptide from the SecYEG. The flagellar rod is a competent drive shaft that transmits torque through the hook to the filament to propel the bacterial locomotion. The distal part of the rod is a helical assembly of FlgG, which shows an obvious sequence similarity with the hook protein. However, the mechanical property of the rod and the hook is quite distinct; the hook is a flexible universal joint, and the rod is a rigid drive shaft. To elucidate the structural basis of the mechanical property of the rod, we crystallized a core fragment of FlgG (FlgG47-227) and solved the structure at 2.0 Å. On the basis of the high resolution X-ray structure and the density ...