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101.
Engineering enzyme specificity by "substrate-assisted catalysis" 总被引:8,自引:0,他引:8
A novel approach to engineering enzyme specificity is presented in which a catalytic group from an enzyme is first removed by site-directed mutagenesis causing inactivation. Activity is then partially restored by substrates containing the missing catalytic functional group. Replacement of the catalytic His with Ala in the Bacillus amyloliquefaciens subtilisin gene (the mutant is designated His64Ala) by site-directed mutagenesis reduces the catalytic efficiency (kcat/Km) by a factor of a million when assayed with N-succinyl-L-Phe-L-Ala-L-Ala-L-Phe-p-nitroanilide (sFAAF-pNA). Model building studies showed that a His side chain at the P2 position of a substrate bound at the active site of subtilisin could be virtually superimposed on the catalytic His side chain of this serine protease. Accordingly, the His64Ala mutant hydrolyzes a His P2 substrate (sFAHF-pNA) up to 400 times faster than a homologous Ala P2 or Gln P2 substrate (sFAAF-pNA or sFAQF-pNA) at pH 8.0. In contrast, the wild-type enzyme hydrolyzes these three substrates with similar catalytic efficiencies. Additional data from substrate-dependent pH profiles and hydrolysis of large polypeptides indicate that the His64Ala mutant enzyme can recover partially the function of the lost catalytic histidine from a His P2 side chain on the substrate. Such "substrate-assisted catalysis" provides a new basis for engineering enzymes with very narrow and potentially useful substrate specificities. These studies also suggest a possible functional intermediate in the evolution of the catalytic triad of serine proteases. 相似文献
102.
Gage KS McAfee JR Carter DA Ecklund WL Riddle AC Reid GC Balsley BB 《Science (New York, N.Y.)》1991,254(5039):1771-1773
Measurement from Christmas Island (2 degrees N, 157 degrees W) of long-term mean vertical motions in the tropical atmosphere using very-high-frequency wind-profiling Doppler radar show that there is a transition from downward motion in the free troposphere to upward motion in the upper troposphere and lower stratosphere. The observations in the free troposphere are consistent with a balance between adiabatic and diabatic heating and cooling rates in a clear atmosphere. Comparison of the results at Christmas Island during El Ni?o and non-El Ni?o conditions with earlier results obtained for stratiform rain conditions over Pohnpei, Federated States of Micronesia, show that cirrus clouds in the vicinity of the tropopause likely play an important role in determining the sense and magnitude of vertical motions in this region. These results have implications for the exchange of mass between the troposphere and stratosphere over the tropics. 相似文献
103.
Wang Z Carter JA Lagutchev A Koh YK Seong NH Cahill DG Dlott DD 《Science (New York, N.Y.)》2007,317(5839):787-790
At the level of individual molecules, familiar concepts of heat transport no longer apply. When large amounts of heat are transported through a molecule, a crucial process in molecular electronic devices, energy is carried by discrete molecular vibrational excitations. We studied heat transport through self-assembled monolayers of long-chain hydrocarbon molecules anchored to a gold substrate by ultrafast heating of the gold with a femtosecond laser pulse. When the heat reached the methyl groups at the chain ends, a nonlinear coherent vibrational spectroscopy technique detected the resulting thermally induced disorder. The flow of heat into the chains was limited by the interface conductance. The leading edge of the heat burst traveled ballistically along the chains at a velocity of 1 kilometer per second. The molecular conductance per chain was 50 picowatts per kelvin. 相似文献
104.
Carter LJ 《Science (New York, N.Y.)》1966,153(3742):1364-1366
105.
106.
Carter LJ 《Science (New York, N.Y.)》1967,155(3760):306-307
107.
Carter LJ 《Science (New York, N.Y.)》1977,197(4300):237-240
108.
O'Connell MJ Bachilo SM Huffman CB Moore VC Strano MS Haroz EH Rialon KL Boul PJ Noon WH Kittrell C Ma J Hauge RH Weisman RB Smalley RE 《Science (New York, N.Y.)》2002,297(5581):593-596
Fluorescence has been observed directly across the band gap of semiconducting carbon nanotubes. We obtained individual nanotubes, each encased in a cylindrical micelle, by ultrasonically agitating an aqueous dispersion of raw single-walled carbon nanotubes in sodium dodecyl sulfate and then centrifuging to remove tube bundles, ropes, and residual catalyst. Aggregation of nanotubes into bundles otherwise quenches the fluorescence through interactions with metallic tubes and substantially broadens the absorption spectra. At pH less than 5, the absorption and emission spectra of individual nanotubes show evidence of band gap-selective protonation of the side walls of the tube. This protonation is readily reversed by treatment with base or ultraviolet light. 相似文献
109.
Carter LJ 《Science (New York, N.Y.)》1968,160(3827):513-517
110.