MEKANISME TRANSLOKASI POLIMERISASI KLENOW-LIKE DNA POLIMERASE I ITB-1 MELALUI PENDEKATAN TARGETED MOLECULAR DYNAMICS

• Main Author: Timotius, Feidy
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/35432
• Institution: Institut Teknologi Bandung

Genes of DNA polymerase I ITB-1 is derived from Geobacillus thermoleovorans bacteria isolated from hot springs Cimanggu. Gene products of DNA Pol I ITB-1 is DNA polymerase I, which has optimum activity at 338 K and at pH 7.4.This enzyme has three important domain, namely domain polymerase 5 '? 3', exonuklease 3'?5' and exonuklease 5'?3'. But in previous research domain 5'?3' eliminated proteolysis with subtilisin protease enzyme and the remaining two domains is called the Klenow fragment. Various studies have been conducted to study this enzyme, starting from the characterization of genes to study the physical interaction between DNA Pol I with the substrate, but studies on the mechanism of this enzyme catalyzed reaction has not been done. Results of previous studies that have been made on several enzyme DNA polymerase, it is known that catalysis of polymerization reactions take place in two stages, namely the binding of dNTP and translocation. Research on the mechanism of translocation is not seintensif research on the mechanism of dNTP binding. This is due to this phase take place quickly (microsecond scale), making it difficult to observe with the experiment although the translocation is the rate determining step (slowest stage), and also difficult to observe with conventional molecular dynamics simulation approach because it takes too long.However, the mechanism of translocation will be more easily learned with a simulation approach because of the ongoing process can be observed more closely at the atomic scale. Therefore, the necessary simulation techniques that can speed up the process of translocation without altering the mechanism. One of the simulation techniques that can be used to overcome this problem is a set of targeted molecular dynamics (TMD). TMD accelerate the process with the help of an external force given in certain parts of the molecule so that it can move faster to achieve the targets set. In this way, the time required to observe the translocation process can be speeded up to the time span that may be achieved by simulation, which is within the range of nano seconds. To perform TMD simulations, the initial structures were prepared from the crystal structure of DNA Pol I from Bacillus stearothermophilus, which has a high homology with DNA Pol I ITB-1. Crystal structures of this enzyme is derived from the protein data bank with access code: 3EYZ (open conformation) and 3EZ5 (closed conformation). The two initial structures is further mutated to have a primary structure identical to the DNA Pol I ITB-1. Amino acids mutated in the two initial structures are D314V, E456A, K505E, G512R, A558S, Y710F, and H823R. The modified strutures then was validated by comparing the value of the beta factor from the simulation data with that of the crystallization data. The results showed that both structures have a similar pattern, thereby the modified structure can be used in subsequent simulation phase. The next stage was to conduct TMD simulations at the optimum temperature of enzyme, which is at 338 K. From the TMD simulation results, it is known that the translocation process begins with the opening of the structure of DNA polymerase I from a closed to open conformation (reopening) followed by the release phase of pyrophosphate ions to the solution. The results showed that the reopening phase is initiated by loss of hydrogen bonds between Arg702 and pyrophosphate ions are interspersed with intermediate stages that take place in the interval 600-800 ps. From the analysis of free-energy change during the process of reopening, shows that this process is not a spontaneous stage as indicated by ?G value of +30.62 kcal / mol. Final stages of post-reaction catalytic mechanism is the process of releasing pyrophosphate ions into solution. From the simulation results of TMD, at this stage pyrophosphate ion will experience a rotation of 180o. During spinning, the pyrophosphate ion will be assisted by Ser655, Gln656, Lys660 and Lys863 residues, which then released into the solution. From the study of free-energy change for dissociation of pyrophosphate ions, this phase has a ?G value of +21.22 kcal/mol, so this stage is non spontaneous stage. Based on the analysis of free energy changes during the simulation, changes in conformation (reopening) and the release of pyrophosphate ions will pass some energy barrier. The largest energy barrier was at the stage of reopening. Therefore, this stage is the rate determining step of the overall DNA polymerization reaction.