Circular dichroism assay for decarboxylation of optically pure amino acids: application to ornithine decarboxylase.
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.
Brooks
H B
HB
Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas, 75235-9041, USA.
Phillips
M A
MA
D002460:Cell Line; D002942:Circular Dichroism; D003653:Decarboxylation; D007700:Kinetics; D009952:Ornithine / Q000378:metabolism*; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D011700:Putrescine / Q000378:metabolism; D013237:Stereoisomerism
D009952:Ornithine; D009955:Ornithine Decarboxylase; D011700:Putrescine