Quantifying enzyme activity in living cells.

ABSTRACT: For over a century, enzymatic activity has been studied in vitro, assuming similar activity in the crowded cellular milieu. Here, we determined in real time the catalytic activity of TEM1-β-lactamase inside living cells and compared the values to those obtained in vitro We found the apparent in vivo catalytic efficiency, k_cat/K_m , to be lower than in vitro, with significant cell-to-cell variability. Surprisingly, the results show that inside the cell the apparent catalytic efficiency decreases, and K_m increases with increasing enzyme concentration. To rationalize these findings, we measured enzyme and substrate diffusion rates in the cell and found the latter to be slower than expected. Simulations showed tha

SUBMITTER: Zotter A 

PROVIDER: S-EPMC5612114 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature