Correlation between Catalytic Efficiency and the Transcription Read-Out in Chemical Complementation: A General Assay for Enzyme Catalysis
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- 作者：Debleena Sengupta ; Hening Lin ; Shalom D. Goldberg ; Jacqueline J. Mahal ; and Virginia W. Cornish
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：March 30, 2004
- 年：2004
- 卷：43
- 期：12
- 页码：3570 - 3581
- 全文大小：196K
- 年卷期：v.43,no.12(March 30, 2004)
- ISSN：1520-4995
文摘
High-throughput assays for enzyme catalysis that can be applied to a broad range of chemicalreactions are key to advances in directed evolution and proteomics. Recently, we reported such a generalassay, chemical complementation, which links enzyme catalysis to reporter gene transcription in vivousing the yeast three-hybrid assay. In this proof-of-principle experiment, it was shown that a wild-type
-lactamase enzyme could be isolated from a pool of inactive mutants using a lacZ screen. Ideally, however,such an assay should be able to distinguish enzymes based on their catalytic activity. Thus, here, we setout to determine if the catalytic efficiency of an enzyme variant does in fact correlate with its level oftranscription activation in the chemical complementation assay. First, the reaction mechanism for thecleavage of the -lactam substrate used in the chemical complementation proof-of-principle experimentwas determined. Then a series of -lactamase variants was designed to span several orders of magnitudein kcat/Km. The activity of each variant was determined both in vitro using purified enzyme and in vivoin the chemical complementation transcription assay. -Lactamase variants spanning three-orders ofmagnitude in kcat/Km could be distinguished in the assay, and the catalytic efficiency of each variantcorrelated with its level of transcription activation in vivo. These results establish the suitability of chemicalcomplementation for the directed evolution of enzymes with improvements in catalytic activity and forprofiling the relative substrate specificities of a group of enzymes in proteomics applications.
-lactamase enzyme could be isolated from a pool of inactive mutants using a lacZ screen. Ideally, however,such an assay should be able to distinguish enzymes based on their catalytic activity. Thus, here, we setout to determine if the catalytic efficiency of an enzyme variant does in fact correlate with its level oftranscription activation in the chemical complementation assay. First, the reaction mechanism for thecleavage of the -lactam substrate used in the chemical complementation proof-of-principle experimentwas determined. Then a series of -lactamase variants was designed to span several orders of magnitudein kcat/Km. The activity of each variant was determined both in vitro using purified enzyme and in vivoin the chemical complementation transcription assay. -Lactamase variants spanning three-orders ofmagnitude in kcat/Km could be distinguished in the assay, and the catalytic efficiency of each variantcorrelated with its level of transcription activation in vivo. These results establish the suitability of chemicalcomplementation for the directed evolution of enzymes with improvements in catalytic activity and forprofiling the relative substrate specificities of a group of enzymes in proteomics applications.