کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1991238 1540987 2016 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Single-molecule enzymology of steroid transforming enzymes: Transient kinetic studies and what they tell us
ترجمه فارسی عنوان
آنزیمولوژی تک مولکول آنزیم های تبدیل کننده استروئیدی: مطالعات جنبشی گذرا و آنچه آنها به ما می گویند
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
چکیده انگلیسی


• Stopped-flow techniques dissect reactions catalyzed by steroid transforming enzymes.
• Ligand binding reactions can identify isomerization steps and new enzyme forms.
• Single turnover experiments isolate the chemistry step.
• Multiple turnover experiments identify burst or linear phase kinetics.
• The technique determines the effects of mutations with precision.

Structure-function studies on steroid transforming enzymes often use site-directed mutagenesis to inform mechanisms of catalysis and effects on steroid binding, and data are reported in terms of changes in steady state kinetic parameters kcat, Km and kcat/Km. However, this dissection of function is limited since kcat is governed by the rate-determining step and Km is a complex macroscopic kinetic constant. Often site-directed mutagenesis can lead to a change in the rate-determining step which cannot be revealed by just reporting a decrease in kcat alone. These issues are made more complex when it is considered that many steroid transforming enzymes have more than one substrate and product. We present the case for using transient-kinetics performed with stopped-flow spectrometry to assign rate constants to discrete steps in these multi-substrate reactions and their use to interpret enzyme mechanism and the effects of disease and engineered mutations. We demonstrate that fluorescence kinetic transients can be used to measure ligand binding that may be accompanied by isomerization steps, revealing the existence of new enzyme intermediates. We also demonstrate that single-turnover reactions can provide a klim for the chemical step and Ks for steroid-substrate binding and that when coupled with kinetic isotope effect measurements can provide information on transition state intermediates. We also demonstrate how multiple turnover experiments can provide evidence for either “burst-phase” kinetics, which can reveal a slow product release step, or linear-phase kinetics, in which the chemical step can be rate-determining. With these assignments it becomes more straightforward to analyze the effects of mutations. We use examples from the hydroxysteroid dehydrogenases (AKR1Cs) and human steroid 5β-reductase (AKR1D1) to illustrate the utility of the approach, which are members of the aldo-keto reductase (AKR) superfamily.

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ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: The Journal of Steroid Biochemistry and Molecular Biology - Volume 161, July 2016, Pages 5–12
نویسندگان
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