Kinetics and thermodynamics of activation of quinoprotein glucose dehydrogenase apoenzyme in vivo and catalytic activity of the activated enzyme in Escherichia coli cells.
Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg(2+). 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 (k(cat)), which were determined to be 2.2x10(3) and 6.8+/-0.8x10(3) 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 (k(f,PQQ)) and with Mg(2+) (k(f,Mg)) were determined to be 3.8+/-0.4x10(4) M(-1).s(-1) and 4. 1+/-0.9 M(-1).s(-1) respectively. Equilibrium constants for the binding of apoenzyme to PQQ and Mg(2+) were determined as the dissociation constants K(d,PQQ(Mg)) and K(d,Mg) to be 1.0+/-0.1 nM and 0.14+/-0.01 mM respectively. The dissociation constants for Ca(2+) were also determined. The holoenzyme, once formed in E. coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg(2+) in solution. EDTA was effective to remove Mg(2+) from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E. coli cells.
Iswantini
D
D
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606 8502, Japan.
Kano
K
K
Ikeda
T
T
D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D012996:Solutions; D013816:Thermodynamics
D012996:Solutions; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase