Recent genomic sequencing of Cryptosporidium muris revealed a phosphagen kinase (PK) homolog. C. muris is a protozoan parasite and the only Cryptosporidium species with a PK, suggesting the gene was acquired via horizontal gene transfer (HGT). Initial sequence alignments suggest that the gene is a creatine kinase enzyme, and the corresponding protein was overexpressed in E. coli and purified to fully characterize its activity. The preferred substrate specificity of the enzyme was tested using 31P-NMR with four guanidino substrates: creatine, arginine, glycocyamine, and taurocyamine in MgATP solutions. Creatine and glycocyamine were identified as the preferred substrates and isothermal titration calorimetry (ITC) was utilized to obtain kinetic constants for the PK. The kinetic values with the creatine substrate were calculated to be Km = 1.95 mM, Vmax = 0.5 uM/s, and Kcat = 30 s-1. Kinetic constants with the glycocyamine substrate were Km = 6.57 mM, Vmax = 0.5 uM/s, and Kcat = 0.5 s-1. A mutant of the enzyme was also created using a truncated N-terminal of the protein sequence and tested utilizing the same characterization methods. 31P-NMR results showed a small amount of activity with only the creatine substrate, but exhibited negligible activity with the ITC. This suggests the truncated N-terminal region plays an important role in enzyme activity.
Biochemistry and Molecular Biology
Piper, Paige, "Characterization of a Creatine Kinase Enzyme From Cryptosporidium Muris" (2013). Senior Independent Study Theses. Paper 331.
creatine kinase, cryptosporidium muris, phosphagen kinase, horizontal gene transfer, evolution
Bachelor of Arts
Senior Independent Study Thesis
© Copyright 2013 Paige Piper