Triose phosphate isomerase (TPI) is a highly-conserved core metabolic enzyme that catalyses the interconversion of D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate during glycolysis and gluconeogenesis. This enzyme is described as ‘perfect’ because the enzyme-catalysed rate of this interconversion is limited only by the rate of diffusion. However, this notion of ‘catalytic perfection’ was arrived at under in vitro conditions, which are not representative of conditions in vivo. We have set out to test whether TPI really is perfect, with respect to its activity under in vivo conditions and its ability to contribute to host cell fitness.
We obtained a plasmid library created by comprehensive codon mutagenesis, which contains variants encoding every possible single amino acid mutation of TPI. The enzymatic activities of these TPI variants in an artificial cytoplasm buffer are being determined via high-throughput assays. The effects of the mutations on cell fitness will be analysed using Phenotype Microarray plates.
Data from several hundred TPI variants indicate a wide distribution of activities, from non-functional to above that of the wild type enzyme. Sequencing and characterisation of individual variants is ongoing. We are also determining the effects of these mutations on cell fitness.
Our activity data confirm that TPI is not a ‘perfect’ enzyme when assayed in artificial cytoplasm. Instead, we have identified mutations that make it even more active. By analysing all possible point mutations, we will ultimately determine the entire activity and fitness landscape of TPI, yielding insights into how natural selection acts upon perfect enzymes.