Improved Catalytic Properties of Halohydrin Dehalogenase by Modification of the Halide-Binding Site
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- 作者：Lixia Tang ; Daniel E. Torres Pazmiñ ; o ; Marco W. Fraaije ; René ; M. de Jong ; Bauke W. Dijkstra ; and Dick B. Janssen
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：May 3, 2005
- 年：2005
- 卷：44
- 期：17
- 页码：6609 - 6618
- 全文大小：223K
- 年卷期：v.44,no.17(May 3, 2005)
- ISSN：1520-4995
文摘
Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes thedehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formationof the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalyticcycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation ofthe X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and betweenthe O
1 atom of Asn176 and N
1 atom of Trp249 could play a role in stabilizing the conformation of thehalide-binding site. The possibility that these hydrogen bonds are important for halide binding and releasewas studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F,which has lost both hydrogen bonds, has a higher catalytic activity (kcat) with two of the three testedsubstrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced kcat value withthese two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalyticefficiency (kcat/Km) was obtained, which is mainly due to an increase of the Km value of the enzyme.Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicatingthat halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescenceexperiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for thewild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that thedisruption of two hydrogen bonds around the halide-binding site increases the rate of halide release andcan enhance the overall catalytic activity of HheC.
1 atom of Asn176 and N1 atom of Trp249 could play a role in stabilizing the conformation of thehalide-binding site. The possibility that these hydrogen bonds are important for halide binding and releasewas studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F,which has lost both hydrogen bonds, has a higher catalytic activity (kcat) with two of the three testedsubstrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced kcat value withthese two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalyticefficiency (kcat/Km) was obtained, which is mainly due to an increase of the Km value of the enzyme.Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicatingthat halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescenceexperiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for thewild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that thedisruption of two hydrogen bonds around the halide-binding site increases the rate of halide release andcan enhance the overall catalytic activity of HheC.