Brooks H B, Phillips M A
Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas, 75235-9041, USA.
Anal Biochem. 1996 Jul 1;238(2):191-4. doi: 10.1006/abio.1996.0274.
A circular dichroism (CD) assay for decarboxylation of optically pure amino acids is described. The viability of this assay is demonstrated using the Trypanosoma brucei ornithine decarboxylase (ODC)-catalyzed reaction of L-ornithine to putrescine and CO2. The results from the CD assay (kcat of 7.5 +/- 0.7 s-1 and Km 230 +/- 60 microM) were identical to the results obtained from the commercially available dye-linked assay which couples CO2 production with NADH oxidation (kcat of 7.3 +/- 0.5 s-1 and Km 320 +/- 30 microM). The CD assay has advantages over the currently used 14CO2 and dye-linked assays since it can be continuously monitored and does not contain additional enzymes. The CD assay will enable the determination of the effects of pH, ionic strength, and D2O on catalysis by ODC. Furthermore, the availability of cuvets with pathlengths from 0.01 to 100 mm provides an effective range for the CD assay from 10 microM to 2.5 M L-ornithine concentration for this assay. This technique should be generally applicable for steady-state analysis of other decarboxylases but is not easily amenable to the analysis of crude enzyme preparations.
本文描述了一种用于光学纯氨基酸脱羧反应的圆二色性（CD）测定法。利用布氏锥虫鸟氨酸脱羧酶（ODC）催化L-鸟氨酸生成腐胺和CO₂的反应，证明了该测定法的可行性。CD测定法的结果（kcat为7.5±0.7 s⁻¹，Km为230±60 μM）与市售的将CO₂生成与NADH氧化偶联的染料连接测定法的结果相同（kcat为7.3±0.5 s⁻¹，Km为320±30 μM）。CD测定法比目前使用的¹⁴CO₂和染料连接测定法具有优势，因为它可以连续监测且不包含额外的酶。CD测定法将能够确定pH、离子强度和重水对ODC催化作用的影响。此外，光程长度从0.01到100 mm的比色皿的可用性为该测定法提供了一个有效的范围，即L-鸟氨酸浓度从10 μM到2.5 M。该技术通常应适用于其他脱羧酶的稳态分析，但不易用于粗酶制剂的分析。
