Measuring enzyme activities under standardized in vivo‐like conditions for systems biology

Abstract

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme-kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme-kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113-7D grown in aerobic glucose-limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h(-1). The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo-like medium containing 300 mM potassium, 50 mM phosphate, 245 mM glutamate, 20 mM sodium, 2 mM free magnesium and 0.5 mM calcium, at a pH of 6.8. The V(max) values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme-specific, optimal conditions.

Integrated multilaboratory systems biology reveals differences in protein metabolism between two reference yeast strains

“…This indicates that the adjustment of the glycolytic flux to fermentative conditions is to a large extent determined by changes in the levels of metabolic intermediates and effectors; that is, flux control is primarily at the metabolic level. This finding is consistent with earlier more detailed studies on glycolysis [18][19][20][21][22] . The metabolome data, however, do not provide any insight into the differences in fluxes between the two strains, as there are very small differences in metabolite levels between the different strains, and most differences indicate slightly higher metabolite concentrations in the YSBN2 strain, which has lower fluxes.…”

“…In connection with the experiment, we also evaluated a new approach to quantify enzyme activities using assay conditions designed to better represent in vivo-like conditions (where the assay medium is designed to mimic the actual intracellular environment, see Methods and Supplementary Methods), as opposed to the typical approach of using optimal conditions for each enzyme (Fig. 3) 19 . We found that with the in vivo-like assays the range of enzyme activities was in the same order of magnitude as the glycolytic flux, which was not observed using traditional analysis of glycolytic enzyme activities.…”

“…Enzyme activities were determined spectrophotometrically in freshly prepared extracts by NAD(P)H-linked assays in either a Novostar, BMG Labtech (VUA), or a TECAN GENios Pro microtiterplate reader (TUD). Standard assays were performed according to Jansen et al 34 In addition, the VUA group also measured activities under a recently proposed in vivo-like assay medium designed to mimic the intracellular environment 19 . All results are presented in µmol min − 1 mg protein − 1 .…”

Tetramers Are the Activation-competent Species of the HOCl-specific Transcription Factor HypT

“…Likewise, the enzyme activity may be different if analyzed in a standard in vitro buffer compared with a buffer that represents the in vivo situation. van Eunen et al (52) analyzed the kinetic parameters of glycolytic and fermentative enzymes of Saccharomyces cerevisiae in a buffer comprising the composition of the yeast cytosol. They showed that some enzymes had largely different activity in the in vivo buffer compared with the enzyme-specific optimum in vitro buffer (52).…”

“…van Eunen et al (52) analyzed the kinetic parameters of glycolytic and fermentative enzymes of Saccharomyces cerevisiae in a buffer comprising the composition of the yeast cytosol. They showed that some enzymes had largely different activity in the in vivo buffer compared with the enzyme-specific optimum in vitro buffer (52). Furthermore, HypT dodecamers are the main species under strong overexpression conditions, whereas small oligomers, as typical for LysR-type transcriptional regulators, prevail in wild-type cells (9).…”