| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
The monooxygenation reaction catalyzed by cytochrome P450 requires two equivalents of proton and electron to cleave dioxygen. This dioxygen scission generates a molecule of water and an oxygen atom that reacts with the substrate, yielding the monooxygenated product. The protons are transferred from the solvent to the active site of the enzyme located interior of the protein. However, how proton is conducted to the active site is still controversial. The objective of this research is to study on the mechanism and pathway of the proton transfer in the d-camphor monooxygenase, cytochrome P450cam, focusing on the entry site of the protons
We have previously proposed the hydrogen-bonding network composed of Thr252-water-Asp251, which extends from the active site to near the protein surface and also proposed this network functions as the proton transfer pathway. The end of the network, Asp251 is within a hydrogen-bonding distance from the surface residue Asp182. This leads us to hypothesize t
… More
hat Asp182 is the proton entry site.
The mutation of Asp182 to Asn slowed the proton-coupled electron transfer from reduced Pdx to oxy-ferrous P450cam (2nd ET) to 7% of the wild type rate. The same mutation, however, did not alter the electron transfer from reduced Pdx to ferric P450cam (1st ET), suggesting that slow proton transfer reduces the rate for 2nd ET. Mutation to Leu reduced the rates for 2nd and 1st ETs to 56 and 77% of the wild type rates, respectively. This small effect of the mutation on the proton transfer contradicts to that of Asn. X-ray structures of the wild type and Asn- and Leu-mutants enzymes demonstrated that the Leu side-chain showed high temperature factors in contrast to those for the side chains of Asn and Asp, both of which formed hydrogen bonds with Arg178 and Arg186. The high temperature factor is deduced to be caused by flexible or mobile side chain, possibly allowing a transient access of water to the protein interior. This possibility was supported by molecular surface analysis of the Leu-mutant with the side chain conformation different from those of Asn and Asp. Therefore, the flexible or mobile side chain explains the fast proton transfer in the Leu-mutant. These results indicate that Asp182 mediates proton transfer from the solvent interface to the active site of P450cam through the previously proposed hydrogen bonded network. Less
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