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|Title:||Kinetics of a Two-Component p-Hydroxyphenylacetate Hydroxylase Explain How Reduced Flavin Is Transferred from the Reductase to the Oxygenase||Contributor(s):||Sucharitakul, J (author); Phongsak, T (author); Entsch, Barrie (author); Svasti, J (author); Chaiyen, P (author); Ballou, DP (author)||Publication Date:||2007||DOI:||10.1021/bi7006614||Handle Link:||https://hdl.handle.net/1959.11/8604||Abstract:||p-Hydroxyphenylacetate hydroxylase (HPAH) from 'Acinetobacter baumannii' catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). HPAH is composed of two proteins: a flavin mononucleotide (FMN) reductase (C₁) and an oxygenase (C₂). C₁ catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH-) for use by C₂ in the hydroxylation reaction. C₁ is unique among the flavin reductases in that the substrate HPA stimulates the rates of both the reduction of FMN and release of FMNH- from the enzyme. This study quantitatively shows the kinetics of how the C₁-bound FMN can be reduced and released to be used efficiently as the substrate for the C₂ reaction; additional FMN is not necessary. Reactions in which O₂ is rapidly mixed with solutions containing C₁-FMNH- and C₂ are very similar to those in which solutions containing O₂ are mixed with one containing the C₂-FMNH- complex. This suggests that in a mixture of the two proteins FMNH- binds more tightly to C₂ and has already been completely transferred to C₂ before it reacts with oxygen. Rate constants for the transfer of FMNH- from C₁ to C₂ were found to be 0.35 and ≥74 s⁻¹ in the absence and presence of HPA, respectively. The reduction of cytochrome c by FMNH- was also used to measure the dissociation rate of FMNH- from C₁. In the absence of HPA, FMNH- dissociates from C1 at 0.35 s⁻¹, while with HPA present it dissociates at 80 s⁻¹; these are the same rates as those for the transfer from C₁ to C₂. Therefore, the dissociation of FMNH- from C₁ is rate-limiting in the intermolecular transfer of FMNH- from C₁ to C₂, and this process is regulated by the presence of HPA. This regulation avoids the production of H₂O₂ in the absence of HPA. Our findings indicate that no protein-protein interactions between C₁ and C₂ are necessary for efficient transfer of FMNH- between the proteins; transfer can occur by a rapid-diffusion process, with the rate-limiting step being the release of FMNH- from C₁.||Publication Type:||Journal Article||Source of Publication:||Biochemistry, 46(29), p. 8611-8623||Publisher:||American Chemical Society||Place of Publication:||Washington, United States of America||ISSN:||0006-2960
|Field of Research (FOR):||060107 Enzymes||Socio-Economic Outcome Codes:||961208 Rehabilitation of Degraded Urban and Industrial Environments||Peer Reviewed:||Yes||HERDC Category Description:||C1 Refereed Article in a Scholarly Journal||Statistics to Oct 2018:||Visitors: 379
|Appears in Collections:||Journal Article|
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