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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.