Studies of the Mechanism of Phenol Hydroxylase: Effect of Mutation of Proline 364 to Serine
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- 作者：Dong Xu ; Cristofer Enroth ; Ylva Lindqvist ; David P. Ballou ; and Vincent Massey
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：November 19, 2002
- 年：2002
- 卷：41
- 期：46
- 页码：13627 - 13636
- 全文大小：245K
- 年卷期：v.41,no.46(November 19, 2002)
- ISSN：1520-4995
文摘
An active site residue in phenol hydroxylase (PHHY), Pro364, was mutated to serine toinvestigate its role in enzymatic catalysis. In the presence of phenol, the reaction between the reducedflavin of P364S and oxygen is very fast, but only 13% of the flavin is utilized to hydroxylate the substrate,compared to nearly 100% for the wild-type enzyme. The oxidative half-reaction of PHHY using m-cresolas a substrate is similarly affected by the mutation. Pro364 was suggested to be important in stabilizingthe transition state of the oxygen transfer step by forming a hydrogen bond between its carbonyl oxygenand the C4a-hydroperoxyflavin [Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J.(2000) J. Am. Chem. Soc. 122, 8728-8738]. The P364S mutation may weaken this interaction by increasingthe flexibility of the peptide chain; hence, the transition state would be destabilized to result in a decreasedlevel of hydroxylation of phenol. However, when the oxidative half-reaction was studied using resorcinolas a substrate, the P364S mutant form was not significantly different from the wild-type enzyme. Therate constants for all the reaction steps as well as the hydroxylation efficiency (coupling between NADPHoxidation and resorcinol consumption) are comparable to those of the wild-type enzyme. It is suggestedthat the function of Pro364 in catalysis, stabilization of the transition state, is not as important in thereaction with resorcinol, possibly because the position of hydroxylation is different with resorcinol thanwith phenol and m-cresol.