Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Measuring the effects of mutations to non-conserved amino acids in lactate dehydrogenase Pohl, Logan E.1, Allan Ayella1,2, and Moriah R. Beck1 1 Chemistry, Wichita State University 2 Natural Sciences, McPherson College Background: Lactate dehydrogenase (LDH), an enzyme is found in cells from nearly all organisms, catalyzes the interconversion of lactate and pyruvate. Although this protein has a highly conserved sequence, it is known to have several non-conserved amino acids that vary between species. Research has suggested that substitutions at non-conserved positions behave more like rheostats in modulating protein function incrementally, whereas substitutions at conserved positions result in “toggle switching” thereby turning activity “on” or “off.” Methods: We first compared protein sequences and structures, and then generated sitedirected mutations at sites that were not conserved among closely related species. These mutated proteins were purified so that we could compare the enzymatic function and stability of the mutant protein to wild-type LDH. CD was used to monitor secondary structure loss during thermal denaturation. Enzyme kinetic parameters were obtained from a kinetic assay that measures formation of NADH. Results: Thermal denaturation curves revealed significant shifts in the melting temperature, indicating differences in the stability for the mutations when compared to the wild type. Kinetic parameters only varied slightly for these mutant proteins. Conclusion: The stability and enzymatic alterations observed indicate the degree these nonconserved regions serve as rheostats for LDH. In the future we hope to apply this knowledge to improve our ability to prediction the effect of genetic alterations that occur in human diseases.