This codebook.txt file was generated on 20201124 by wilsonkm ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset Steady-state coupled enzyme assays using standard peptides of cAMP-dependent protein kinase a catalytic subunit (PKA-C) and its chimeric mutant (PKA-CJ) 2. Author Information Principal Investigator Contact Information Name: Cristina Olivieri Institution: University of Minnesota Email: colivier@umn.edu 3. Date of data collection: July 2018 - October 2020 -------------------------- SHARING/ACCESS INFORMATION -------------------------- 1. Licenses/restrictions placed on the data: CC0 1.0 Universal 2. Links to publications that cite or use the data: This data set are part of the following publication "Defective Internal Allosteric Network Imparts Dysfunctional ATP/Substrate 1 Binding Cooperativity in Oncogenic Chimera of Protein Kinase A", that is under review in Communication Biology journal. 3. Recommended citation for the data: (2020). Steady-state coupled enzyme assays using standard peptides of cAMP-dependent protein kinase a catalytic subunit (PKA-C) and its chimeric mutant (PKA-CJ). Retrieved from the Data Repository for the University of Minnesota, http://hdl.handle.net/11299/217205. --------------------- DATA & FILE OVERVIEW --------------------- 1. File List A. Filename: Kemptide-CREB-KSR1_ActivityAssay_102120.xlsx Short description: Steady-state coupled enzyme assays - raw data B. Filename: Kemptide-CREB-KSR1_ActivityAssay_102120.pzfx Short description: Steady-state coupled enzyme assays - processed data The .pzfx file can viewed by using Prism Viewer, which can be found on graphpad.com. 2. Relationship between files: In A are listed raw data, as intensity vs time, obtianed from the UV spectrometer (Spectromax). The enzymatic assay was performed usig PKA-C and PKA_JC and three different subtrates (Kemptide, CREB and KSR1). In B is reported the processing and fitting of the data in A, using the standard steady-state Michaelis-Menten kinetics equation. File B are also reported all the kinetics parameters for the two enzymes and the three different substrates. 3. Are there multiple versions of the dataset? no -------------------------- METHODOLOGICAL INFORMATION -------------------------- 1. Description of methods used for collection/generation of data: Steady-state coupled-enzyme assays of PKA-C and PKA-JC using Kemptide, CREB- and KRS1-peptide as substrated. The couple-enzyme is a in-vitro assay, commonly used to characterize the kinetic parameters of protein kinases and ATPases. Cook, P. F., Neville, M. E., Jr., Vrana, K. E., Hartl, F. T. & Roskoski, R., Jr. Adenosine cyclic 3',5'-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit. Biochemistry 21, 5794-5799 (1982). The ADP produced by the phosphoryl-transfer reaction catalyzed by target enzyme is used by the Pyruvate kinase (PK)to convert the phosphoenol pyruvate (PEP) to pyruvate+ATP. The pyruvate is then converted to lactate by the Lactic Dehydrogenase (LDH) by the reduction of NADH to NAD+. The deplition of the signal of NADH at 340nm is monitored. 2. Methods for processing the data: The raw data were collected at intensity vs time and coverted to substrate concentation vs rate. The catalytic parameter (Vmax and Km) were obtained from a non-linear fit of the initial velocities to the Michaelis-Menten equation 3. Instrument- or software-specific information needed to interpret the data: The data were acquired using SPECTRAmax 384PLUS UV-spectrometer, equipped with a 96well plate reader. The data were acquired using the instrument software, organized using Microsoft Excel, and analyzed using GraphPad Prim 8.0 4. Standards and calibration information, if appropriate: All the kinetic reaction where conducted in triplicare for each substrate concentration. A blanck reaction (reaction w/o the target enzyme) was also performed in triplicate. 5. Environmental/experimental conditions: All the experiment were acquired at 298 K 6. Describe any quality-assurance procedures performed on the data: 7. People involved with sample collection, processing, analysis and/or submission: Caitlin Walker performed the experiment, processed and analyzed the data Cristina Olivieri submitted the data to the repository ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Kemptide-CREB-KSR1_ActivityAssay_102120.xlsx ----------------------------------------- The .xlsx workbook contains multiple sheets: Ca Kemptide JCa Kemptide Ca CREBtide JCa CREBtide Ca KSR1 JCa KSR1 1. Number of variables: 8 2. Number of cases/rows: 12 3. Missing data codes: Code/symbol Definition Code/symbol Definition 4. Variable List A. Name: [S], uM Description: Substrate concentration used in the assay expressed in micromolar concentration (uM) B. Name: Rate(mAu/min) Description: kinetic rated for the phosphorylation reaction C. Name: Average Rate (mAu/min) Description: Each reaction has been repeated in triplicate. This value is the averege between the three rates calculated from the triplicate D. Name: Accounting for volume Description: accounting the dilution factor E. Name: Accounting for NADH absorbance F. Name: Lineweaver-Burk Plot Description: Linearization plot of the raf extimation of Vmax and Km G. Name: Stdev Description: standard deviation H. Name: Accounting for NADH absorbance Description: Blank value