caa a22 4500 44532550X CHVBK 20180317142719.0 cr unu---uuuuu 170323e20110901xx s 000 0 eng 10.1007/s10858-011-9538-9 doi (NATIONALLICENCE)springer-10.1007/s10858-011-9538-9 Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis [Elektronische Daten] [Alexander Greenwood, Monique Rogals, Soumya De, Kun Lu, Evgenii Kovrigin, Linda Nicholson] The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, $$ k_{cat}^{cis} $$ and apparent Michaelis constants, $$ K_{M}^{App} $$ . By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide 13C-1H constant time HSQC spectra to determine $$ k_{cat}^{cis} $$ , $$ k_{cat}^{trans} $$ , $$ K_{D}^{cis} $$ , and $$ K_{D}^{trans} $$ for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis]=3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme. Springer Science+Business Media B.V., 2011 Proyl isomerase nationallicence Pin1 nationallicence Lineshape analysis nationallicence Isomerization nationallicence Proline nationallicence APP nationallicence Greenwood Alexander Department of Molecular Biology and Genetics, Cornell University, 14853, Ithaca, NY, USA aut Rogals Monique Department of Molecular Biology and Genetics, Cornell University, 14853, Ithaca, NY, USA aut De Soumya Department of Molecular Biology and Genetics, Cornell University, 14853, Ithaca, NY, USA aut Lu Kun Cancer Biology Program and Biology of Aging Program, Beth Israel Deaconess Medical Center, Harvard Medical School, 30 Brookline Avenue, CLS 0408, 02215, Boston, MA, USA aut Kovrigin Evgenii Department of Biochemistry, Medical College of Wisconsin, 53226, Milwaukee, WI, USA aut Nicholson Linda Department of Molecular Biology and Genetics, Cornell University, 14853, Ithaca, NY, USA aut Journal of Biomolecular NMR Springer Netherlands 51/1-2(2011-09-01), 21-34 0925-2738 51:1-2<21 2011 51 10858 https://doi.org/10.1007/s10858-011-9538-9 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9538-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Greenwood Alexander Department of Molecular Biology and Genetics, Cornell University, 14853, Ithaca, NY, USA aut NATIONALLICENCE 700 1- Rogals Monique Department of Molecular Biology and Genetics, Cornell University, 14853, Ithaca, NY, USA aut NATIONALLICENCE 700 1- De Soumya Department of Molecular Biology and Genetics, Cornell University, 14853, Ithaca, NY, USA aut NATIONALLICENCE 700 1- Lu Kun Cancer Biology Program and Biology of Aging Program, Beth Israel Deaconess Medical Center, Harvard Medical School, 30 Brookline Avenue, CLS 0408, 02215, Boston, MA, USA aut NATIONALLICENCE 700 1- Kovrigin Evgenii Department of Biochemistry, Medical College of Wisconsin, 53226, Milwaukee, WI, USA aut NATIONALLICENCE 700 1- Nicholson Linda Department of Molecular Biology and Genetics, Cornell University, 14853, Ithaca, NY, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 51/1-2(2011-09-01), 21-34 0925-2738 51:1-2<21 2011 51 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer