Evolutionary study of the phosphagen kinase enzyme family probing substrate specificity and cooperativity in software derived ancestral proteins

Eric Johnson, The College of Wooster


Changes in protein structure and function are major components of species evolution and thus, to better understand proteins evolution is to better understand the evolution of life. In order to elucidate the mechanisms by which proteins evolve, we must probe the current diversity in protein families in hopes to gain insight regarding their evolutionary history. The phosphagen kinase (PK) enzyme family provides an excellent model for the study of protein evolution for its great diversity in biochemical properties and wide dispersal among multi-cellular organisms. PKs catalyze the reversible transfer of the gamma phosphate off of ATP onto a guanidino group on an acceptor molecule. These phosphagen compounds can be used as energy currency throughout the cell in times of need. Recently, a study was completed implementing four ancestral protein sequences (AnPK42, AnPK46, AnPK50, and AnPK52) generated using the computer program ANCESCON (Van Houten, unpublished). The four ancestral gene sequences were synthesized and cloned into plasmids, which were transformed into competent cells, then over-expressed and purified in Luria Bertani broth. Using HPLC, ITC and bioinformatics, Van Houten predicted AnPK42 to be a CK, while AnPK50 and AnPK52 to be TKs. A complete kinetic global analysis of AnPK42 and AnPK50 was performed, while only partial completion was achieved for AnPK52. No kinetic or substrate data was produced for AnPK46. The aim of this study was to better elucidate the evolutionary pathway of phosphagen kinases by expanding on the previous results and to probe for cooperativity in AnPK42. 31P-NMR and ITC results show AnPK46's preferred substrate is taurocyamine, but activity is very low. Based in part upon the results obtained in this study, a new evolutionary model is presented.


© Copyright 2012 Eric Johnson