昇温熱分解¹⁴C法による堆積物年代推定と北極海イベント層の層位学的研究 [論文内容及び審査の要旨]

The role of protein dynamics in the reaction catalyzed by dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) has been examined by enzyme isotope substitution (¹⁵N, ¹³C, ²H). In contrast to all other enzyme reactions investigated previously, including DHFR from Escherichia coli (EcDHFR), for which isotopic substitution led to decreased reactivity, the rate constant for the hydride transfer step is not affected by isotopic substitution of TmDHFR. TmDHFR therefore appears to lack the coupling of protein motions to the reaction coordinate that have been identified for EcDHFR catalysis. Clearly, dynamical coupling is not a universal phenomenon that affects the efficiency of enzyme catalysis

Thermal Adaptation of Dihydrofolate Reductase from the Moderate Thermophile <i>Geobacillus stearothermophilus</i>

The thermal melting temperature of dihydrofolate reductase from <i>Geobacillus stearothermophilus</i> (BsDHFR) is ∼30 °C higher than that of its homologue from the psychrophile <i>Moritella profunda</i>. Additional proline residues in the loop regions of BsDHFR have been proposed to enhance the thermostability of BsDHFR, but site-directed mutagenesis studies reveal that these proline residues contribute only minimally. Instead, the high thermal stability of BsDHFR is partly due to removal of water-accessible thermolabile residues such as glutamine and methionine, which are prone to hydrolysis or oxidation at high temperatures. The extra thermostability of BsDHFR can be obtained by ligand binding, or in the presence of salts or cosolvents such as glycerol and sucrose. The sum of all these incremental factors allows BsDHFR to function efficiently in the natural habitat of <i>G. stearothermophilus</i>, which is characterized by temperatures that can reach 75 °C

Effect of Dimerization on Dihydrofolate Reductase Catalysis

Dihydrofolate reductase (DHFR) from the hyperthermophile <i>Thermotoga maritima</i> (TmDHFR) forms a very stable homodimer, while DHFRs from other organisms are monomers. We investigated the effect of dimerization on DHFR catalysis by preparing a dimeric variant, Xet-3, of DHFR from <i>Escherichia coli</i> (EcDHFR). Introducing residues located at the TmDHFR dimer interface into EcDHFR increases the melting temperature to ∼60 °C, approximately 9 °C higher than that measured for EcDHFR. The steady-state and pre-steady-state rate constants measured for Xet-3 were similar to those of dimeric TmDHFR but significantly lower than those of the parent EcDHFR. This reduction in the degree of catalytic competence is likely a consequence of the loss of flexibility of catalytically important loop regions of EcDHFR on dimerization and a compromise of the electrostatic environment of the active site. In contrast, the reduced catalytic ability of TmDHFR relative to that of EcDHFR is not simply a consequence of reduced loop flexibility in the dimeric enzyme. Our studies demonstrate that EcDHFR is not a good model for understanding the properties of other DHFRs, including TmDHFR

Loop Interactions during Catalysis by Dihydrofolate Reductase from <i>Moritella profunda</i>

Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from <i>Moritella profunda</i> (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the well-studied DHFR from <i>Escherichia coli</i> (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR

NMR Solution Structure of a Photoswitchable Apoptosis Activating Bak Peptide Bound to Bcl-x_L

The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-x_L. This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states

Increased Dynamic Effects in a Catalytically Compromised Variant of <i>Escherichia coli</i> Dihydrofolate Reductase

Isotopic substitution (¹⁵N, ¹³C, ²H) of a catalytically compromised variant of <i>Escherichia coli</i> dihydrofolate reductase, EcDHFR-N23PP/S148A, has been used to investigate the effect of these mutations on catalysis. The reduction of the rate constant of the chemical step in the EcDHFR-N23PP/S148A catalyzed reaction is essentially a consequence of an increase of the quasi-classical free energy barrier and to a minor extent of an increased number of recrossing trajectories on the transition state dividing surface. Since the variant enzyme is less well set up to catalyze the reaction, a higher degree of active site reorganization is needed to reach the TS. Although millisecond active site motions are lost in the variant, there is greater flexibility on the femtosecond time scale. The “dynamic knockout” EcDHFR-N23PP/S148A is therefore a “dynamic knock-in” at the level of the chemical step, and the increased dynamic coupling to the chemical coordinate is in fact detrimental to catalysis. This finding is most likely applicable not just to hydrogen transfer in EcDHFR but also to other enzymatic systems

Differences in Functional Significance of Intra-Left Ventricular Vortex on Energy Efficiency among Normal, Dilated, and Hypertrophied Hearts [an abstract of dissertation and a summary of dissertation review]

配架番号：253

The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium <i>Moritella profunda</i> (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from <i>Escherichia coli</i> (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR