http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
Engineering of Substrate Specificity of Horse Liver Alcohol Dehydrogenase on Steroids
박두홍 한국공업화학회 1992 한국공업화학회 연구논문 초록집 Vol.1992 No.0
The E and S isoenzymes of horse liver alcohol dehydrogenase differ by 10 amino acid residues, but only the S isoenzyme is active on 3β-hydroxysteroids. The cDNAs for these isoenzymes were cloned and expressed in E. coli. The steroid activity of the S isoenzyme was correlated to structure of the isoenzymes by characterizing a series of chimeric enzymes, which could represent intermediates in the evolution of catalytic activity. Deletion of Asp-115 from the E isoenzyme created the E/D115 Δ enzyme that is active on steroids. The deletion alters the substrate binding pocket by moving Leu-116, which sterically hinders binding of steroids in the E isoenzyme. A chimeric enzyme (ESE) that has four changes in or near the substrate binding pocket (T94I/R101S/F110L/D115 Δ) was 15-30-fold more catalytically efficient (V/Km) on uncharged steroids than was the E/D115 Δ enzyme. Molecular modeling suggests that the substitutions at residues 94 and 110 indirectly affect the activity on steroids. ESE enzyme was 6-fold more active than the S isoenzyme on neutral steroids, due to substitutions not in the substrate binding pocket. The K366E and the Q17E/A43T/A59T substitutions in the S isoenzyme gave 2-ioid increases in V/Km on steroids, which together can account for the changes observed with the ESE enzyme. The enzymes that are active on steroids did not bind 2,2,2-trifluoroethanol as tightly and were catalytically less efficient than the E isoenzyme with small alcohols. However, these enzymes were two to three and four to five orders of magnitude more efficient with 1-hexanol and 5β-androstane-3β, 17β-diol, respectively, than with ethanol. These results demonstrate that several residues not directly participating in substrate binding or chemical catalysis contribute to catalytic efficiency.
An Experimental Study on the Coupled Reaction System Comprised of Chromatophore and NAD Kinase
박두홍,최차용,Park, Doo-H.,Choi, Cha-Y. 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.1
광 에너지 흡수 시스템으로서의 크로마토포아를 에너지 소비 시스템인 NAD Kinase에 공역시켜 ATP를 중간 에너지 운반체로 하는 NADP 생산을 수행하였다. 이 공역 시스템과 NAD Kinase 만을 사용하는 단일 시스템을 비교해 본 결과 사용한 초기 ATP 농도에 따라 다르기는 하나 공역 시스템의 생산성이 단일 시스템에 비해 30% 내지 80% 높은 것을 알 수 있었다. 그러나 초기 ATP 농도가 너무 높을 때는 공역 시스템의 생산성이 단일 시스템의 그것 보다 도리어 못한 결과를 보여주었다. 사용한 크로마토포아 농도에 있어서도 공역 시스템의 생산성을 최대로 해주는 최적 농도가 있음을 알게 되었다. 이와같은 최적 크로마토포아 농도의 존재는 크로마토포아 자체에 의한 광 차폐효과 때문인 것으로 사료된다. 이 광 차폐효과는 크로마토포아 단독 시스템에 있어서 직경이 다른 두개의 시험관 내에서 조사되었다. 이 단독 시스템에 의한 ADP로부터의 ATP 생산에 있어서도 생산성을 최대로 해주는 최적 크로마토포아 농도가 있었으며 이 최적농도는 반응기의 크기가 커질수록 줄어 들었다. 크로마토포아의 반응기에의 도입시간에 따른 NADP 생산성 변화도 조사해 보았던 바 최적도입 시간이 존재함을 알 수 있었다. Chromatophore as a light-energy-absorbing system was coupled with an energy-consuming system, NAD kinase, to produce NADP from NAD using ATP as the intermediate energy carrier. The enhancement in the NADP production of the coupled system as compared with the single system containing NAD kinase alone ranged between 30% and 80% depending upon the initial concentration of ATP used in both systems. However, the use of too high an initial concentration of ATP caused a worse NADP production of the coupled system than the single system. There was an optimal concentration of chromatophore at which the NADP production of the coupled system was maximized. This seemed to be due to the shielding effect of the chromatophores themselves. This shielding effect was also investigated with the single reaction system now comprising chromatophore alone in two reaction test tubes of different diameters. The specific ATP production from ADP showed a maximum point when plotted against the chromatophore concentration and this optimal chromatophore concentration decreased with increasing size of the reaction vessel. Dependence of the NADP production on the time of chromatophore introduction into the reaction system was investigated. An optimal chromatophore introduction time was found to exist for the maximum production of NADP.