Kinetics and thermodynamics of activation of quinoprotein glucose dehydrogenase apoenzyme in vivo and catalytic activity of the activated enzyme in Escherichia coli cells

Abstract

Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg2+. Catalytic behaviour of the E. coli cells with the holoenzyme is characterized by a Michaelis–Menten-type equation with a catalytic constant of the cell and apparent Michaelis constants for d-glucose and an artificial electron acceptor added to the E. coli suspension. The catalytic constant is expressed as the product of the number of molecules of the enzyme contained in an E. coli cell (z) and the catalytic constant of the enzyme (kcat), which were determined to be 2.2×103 and 6.8±0.8×103s-1 (phenazine methosulphate as an electron acceptor) respectively. Kinetics of the in vivo holoenzyme formation can be followed by an enzyme-electrochemical method developed by us. The rate constants for the reactions of apoenzyme with PQQ (kf,PQQ) and with Mg2+ (kf,Mg) were determined to be 3.8±0.4×104 M-1·s-1 and 4.1±0.9M-1·s-1 respectively. Equilibrium constants for the binding of apoenzyme to PQQ and Mg2+ were determined as the dissociation constants Kd,PQQ(Mg) and Kd,Mg to be 1.0±0.1nM and 0.14±0.01mM respectively. The dissociation constants for Ca2+ were also determined. The holoenzyme, once formed in E. coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg2+ in solution. EDTA was effective to remove Mg2+ from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E. coli cells.