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Why in vivo may not equal in vitro – new effectors revealed by measurement of enzymatic activities under the same in vivo ‐like assay conditions
Author(s) -
GarcíaContreras Rodolfo,
Vos Paul,
Westerhoff Hans V.,
Boogerd Fred C.
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12007
Subject(s) - phosphoglycerate kinase , biochemistry , phosphoglycerate mutase , glutamate dehydrogenase , in vivo , azaserine , enzyme , triosephosphate isomerase , aldolase a , glutamine synthetase , phosphofructokinase , biology , glycolysis , dehydrogenase , glutamine , fructose bisphosphate aldolase , glyceraldehyde 3 phosphate dehydrogenase , phosphoglucomutase , glutaminase , glutamine amidotransferase , glutamate receptor , amino acid , receptor , microbiology and biotechnology
Does the understanding of the dynamics of biochemical networks in vivo , in terms of the properties of their components determined in vitro , require the latter to be determined all under the same conditions? An in vivo ‐like assay medium for enzyme activity determination was designed based on the concentrations of the major ionic constituents of the E scherichia coli cytosol: K + , N a + , M g 2+ , phosphate, glutamate, sulfate and Cl − . The maximum capacities ( V max ) of the extracted enzymes of two pathways were determined using both this in vivo ‐like assay medium and the assay medium specific for each enzyme. The enzyme activities differed between the two assay conditions. Most of the differences could be attributed to unsuspected, pleiotropic effects of K + and phosphate. K + activated some enzymes (aldolase, enolase and glutamate dehydrogenase) and inhibited others (phosphoglucose isomerase, phosphofructokinase, triosephosphate isomerase, glyceraldehyde 3‐phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase), whereas phosphate inhibited all glycolytic enzymes and glutamine synthetase but only activated glutamine 2‐oxoglutarate amidotransferase. Neither a high glutamate concentration, nor macromolecular crowding affected the glycolytic or nitrogen assimilation enzymes, other than through the product inhibition of glutamate dehydrogenase by glutamate. This strategy of assessing all pathway enzymes kinetically under the same conditions may be necessary to avoid inadvertent differences between in vivo and in vitro biochemistry. It may also serve to reveal otherwise unnoticed pleiotropic regulation, such as that demonstrated in the present study by K + and phosphate.