Proton-transfer mechanism of pyridoxal enzymes studied by 3D-free energy profile
| Project/Area Number |
13680697
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Osaka Medical College |
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Principal Investigator |
HAYASHI Hideyoshi Osaka Medical College Faculty of Medicine Associate Professor, 医学部, 助教授 (00183913)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | proton-transfer / pKa / catalytic-mechanism / aminotransferase / pyridoxal-phosphate / strain / free-energy-profile / energy-barrier / 自由エネルギー |
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| Research Abstract |
The detailed mechanism of proton transfer from the substrate to the coenzyme Schiff base in aspartate aminotransferase (AAT) and the structural basis for the driving force have been elucidated through 3D-free energy analysis. The use of V393F mutation, which perturbs the formation of the lid over the active site, clarified that the unfavorable interaction between Gly38 and the unprotonated Schiff base elevates the free energy level of the Michaelis complex, thereby increasing the k_<cat> value. Thus, the classical explanation for the increase in the Schiff base pK_a has been found to be nothing more than a reflection of the destabilization of the Michaelis complex.
Analysis of the reaction of AAT with C5 substrates revealed that the C5 substrate, unlike the C4 substrate such as aspartate, take an extended conformation in the open form of AAT. The decrease in the K_m value and unchanged k_<cat> value of the V39L mutant enzyme were assessed based on the 3D-free energy analysis in which the conformation of AAT was taken as the third axis of the 3D profile. The analysis showed that AAT takes a strategy to increase the k_<cat> value toward the C5 substrate at the expense of K_m by destabilizing the open form of the Michaelis complex.
The present study clearly showed the effectiveness of the 3D-free energy analysis. The method is considered to be applicable to many aspects of the enzyme catalysis, if an appropriate coordinate is taken as the third axis.
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Report
(3 results)
Research Products
(36 results)
