(S)-Mandelate Dehydrogenase from Pseudomonas putida: Mutations of the Catalytic Base Histidine-274 and Chemical Rescue of Activity
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- 作者：Isabelle E. Lehoux and Bharati Mitra
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 3, 1999
- 年：1999
- 卷：38
- 期：31
- 页码：9948 - 9955
- 全文大小：91K
- 年卷期：v.38,no.31(August 3, 1999)
- ISSN：1520-4995
文摘
(S)-Mandelate dehydrogenase from Pseudomonas putida, an FMN-dependent 
-hydroxy aciddehydrogenase, oxidizes (S)-mandelate to benzoylformate. The generally accepted catalytic mechanismfor this enzyme involves the formation of a carbanion intermediate. Histidine-274 has been proposed tobe the active-site base that abstracts the substrate -proton to generate the carbanion. Histidine-274 wasaltered to glycine, alanine, and asparagine. All three mutants were completely inactive. The mutants wereable to form adducts with sulfite, though with much weaker affinity than the wild-type enzyme. Bindingof the inhibitor, (R)-mandelate, was not greatly affected by the mutation, unlike that of the substrate,(S)-mandelate, indicating that H274 plays a role in substrate binding. The activity of H274G and, to alesser extent, H274A could be partially restored by the addition of exogenous imidazoles. The maximumrescued activity for H274G with imidazole was ~0.1% of the wild-type value. Saturation kinetics obtainedfor rescued activity suggest that formation of a ternary complex of imidazole, enzyme, and substrate isrequired for catalysis. pH-dependence studies confirm that the free base form of imidazole is the rescueagent. An earlier study of pH profiles of the wild-type enzyme indicated that deprotonation of a residuewith a pKa of 5.5 in the free enzyme was essential for activity (Lehoux, I. E., and Mitra, B. (1999)Biochemistry 38, 5836-5848). Data obtained in this work confirm that the pKa of 5.5 belongs to histidine-274.
-hydroxy aciddehydrogenase, oxidizes (S)-mandelate to benzoylformate. The generally accepted catalytic mechanismfor this enzyme involves the formation of a carbanion intermediate. Histidine-274 has been proposed tobe the active-site base that abstracts the substrate -proton to generate the carbanion. Histidine-274 wasaltered to glycine, alanine, and asparagine. All three mutants were completely inactive. The mutants wereable to form adducts with sulfite, though with much weaker affinity than the wild-type enzyme. Bindingof the inhibitor, (R)-mandelate, was not greatly affected by the mutation, unlike that of the substrate,(S)-mandelate, indicating that H274 plays a role in substrate binding. The activity of H274G and, to alesser extent, H274A could be partially restored by the addition of exogenous imidazoles. The maximumrescued activity for H274G with imidazole was ~0.1% of the wild-type value. Saturation kinetics obtainedfor rescued activity suggest that formation of a ternary complex of imidazole, enzyme, and substrate isrequired for catalysis. pH-dependence studies confirm that the free base form of imidazole is the rescueagent. An earlier study of pH profiles of the wild-type enzyme indicated that deprotonation of a residuewith a pKa of 5.5 in the free enzyme was essential for activity (Lehoux, I. E., and Mitra, B. (1999)Biochemistry 38, 5836-5848). Data obtained in this work confirm that the pKa of 5.5 belongs to histidine-274.