(623p) Control of Enzyme Activity by Introduction of Molecular Recognition Moiety
Regulation of enzyme activity by biding a ligand or protein, such as allosteric enzyme and signal transduction, is common in vivo. This enzyme function is attractive from a view of application for bio reactor, DDS, and bio sensor. Some research groups developed conjugation of enzyme and stimuli responsive polymer to control enzyme activity or specific binding by stimulus such as temperature, pH, and light for affinity separation, microfluidic protein analysis and capture[1-3]. However, in order to control the enzyme activity by molecular recognition, the problem is that how to modify the surface of the enzyme to create the binding site for another ligand and how to control the activity of the enzyme when the ligand is recognized. There are some previous studies controlling enzyme activity by genetic modification or antigen-antibody complex reaction. These approaches utilize conformational change of enzyme with genetic modification or protein-ligand interaction. However, the reaction mechanism is complicated and is mostly difficult to know, thus we must repeat the genetic modification process and change the position of binding site by trial and error approach. The approach for making molecular recognition enzyme in previous researches also lacks versatility.
Our approach to develop novel functional enzyme is that molecular recognition moiety is conjugated only near active pocket of the mutant enzyme. In this research, Cytochrome P450cam was used as enzyme and biotin with spacer arm was used as molecular recognition moiety. This enzyme regulates its activity by utilizing avidin-biotin affinity interaction as the following. In the absence of avidin, there is no steric hindrance and the enzymatic reaction proceeds normally. On the other hand, in the presence of avidin, the avidin that binds to biotin covers the active pocket of the enzyme, then enzyme reaction does not proceed by the inhibition of substrate diffusion. Therefore, the enzyme can control activity itself in response to specific molecule. The advantage of this approach is that we can design the required enzymatic function by changing both enzyme and molecular recognition moiety.
In this study, we firstly fabricated genetically modified P450cam that has one thiol group near its active pocket, abbreviated as 3mD. This mutant has a property that thiol-reacticve pegylated biotin is able to be conjugated only at targeted position. Then, conjugation of 3mD and pegylated biotin with different length of spacer arm was performed. Finally, the concept of this study was proven and the effect of the length of spacer arm on the avidin signal response was also examined.
The activity of 3mD was retained compared to that of wild type P450cam. Furthermore, 3mD kept their tertial structure after genetic modification by measuring absorption peak of ferrous CO-complexed state. This shows that 3mD maintained enough activity without any denaturation. The number of thiol group on the surface of 3mD were investigated. From thiol group determination, about 0.7 mol of thiol groups existed in 1 mol of 3mD, while thiol group on the surface of wild type P450cam was not detected. This result is close to theoretical value of 3mD. These results suggest that 3mD was prepared intendedly.
Then, three kinds of pegylated biotins that have different length of spacer arm were conjugated to 3mD. The length of spacer arms are 39 atoms (biotin-PEG11), 12 atoms (biotin-PEG2), and 2 atoms (biotin-Alkyl2) respectively. This conjugation was confirmed by determining the amount of thiol groups before and after biotinylation and about 70% of thiol groups were successfully biotinylated. Finally, we evaluated each activity of biotinylated 3mD before and after avidin binding. In the case of biotin-PEG11-3mD, which has the longest spacer arm, the activity did not change when avidin binds to biotin. On the other hand, the activity of 3mD with shorter spacer arm, especially biotin-Alkyl2-3mD, dramatically decreased to 60% after avidin binding. The diameter of the active pocket is around 17 angstrom, which is almost the same as the length of spacer arm in biotin-Alkyl2-3mD. The conjugation site exists on the circumference of this pocket. As for biotin-PEG11-3mD, bound avidin was too far from the active pocket of 3mD to cover the pocket. Meanwhile, as the length of spacer arm is shorter, bound avidin get closer to the active pocket, and the avidin appropriately covered the active pocket in biotin-Alkyl2-3mD. We can conclude that we successfully developed novel functional enzyme and optimized its nanostructure.
 A. S. Hoffman, Clinical Chemistry 46, 1478 (2000).
 A. S. Hoffman et al., Journal of Biomedical Materials Research 52, 577 (2000).
 C. D. H. Alarcon, S. Pennadam, C. Alexander, Chemical Society Reviews 34, 276 (2005).
 F. T. K. Lau, A. R. Fersht, Nature 326, 811 (1987).
 C. A. Brennan, K. Christianson, M. A. Lafleur, W. Mandecki, Proceedings of the National Academy of Sciences of the United States of America 92, 5783 (1995).
 S. A. Martinis et al., Biochemistry 35, 14530 (1996).