In vitro turnover numbers do not reflect in vivo activities of yeast enzymes.

Abstract

Turnover numbers (<i>k</i>_cat values) quantitatively represent the activity of enzymes, which are mostly measured in vitro. While a few studies have reported in vivo catalytic rates (<i>k</i>_app values) in bacteria, a large-scale estimation of <i>k</i>_app in eukaryotes is lacking. Here, we estimated <i>k</i>_app of the yeast <i>Saccharomyces cerevisiae</i> under diverse conditions. By comparing the maximum <i>k</i>_app across conditions with in vitro <i>k</i>_cat we found a weak correlation in log scale of <i>R</i>² = 0.28, which is lower than for <i>Escherichia coli</i> (<i>R</i>² = 0.62). The weak correlation is caused by the fact that many in vitro <i>k</i>_cat values were measured for enzymes obtained through heterologous expression. Removal of these enzymes improved the correlation to <i>R</i>² = 0.41 but still not as good as for <i>E. coli</i>, suggesting considerable deviations between in vitro and in vivo enzyme activities in yeast. By parameterizing an enzyme-constrained metabolic model with our <i>k</i>_app dataset we observed better performance than the default model with in vitro <i>k</i>_cat in predicting proteomics data, demonstrating the strength of using the dataset generated here.