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 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.

Electrochemical Glucose Sensors and Their Applications in Diabetes Management

“…The purpose of their mutation and expression in different organisms was to increase enzyme yield, facilitate enzyme purification, increase specific activity, improve the enzyme stability, and enhance selectivity for glucose. [24][25][26][27][28][29][30][31][32][33][34][35] The two enzyme families differ in their redox potentials, the strengths of the bonds between their protein-devoid apoenzymes and their cofactors, their cosubstrates, their turnover rates, their Michaelis constants (K m ), and their selectivity for glucose.…”

Bioelectrochemical sensor based on PQQ-dependent glucose dehydrogenase

“…As compared to NAD-dependent glucose dehydrogenase that uses soluble coenzyme NAD, PQQ-GDH contains a relatively tightly bound PQQ cofactor. The equilibrium constant for the binding of PQQ to soluble apo-GDH isolated from citosol of Escherichia coli of (1 ± 0.1) × 10 −9 M has been reported [3].…”

“…1-Methoxyphenazonium as an electron shuttle between PQQ-GDH and electrode has been used as a part of hybridization event transducer in DNA sensor design [5]. NMP has been shown to be an efficient electron shuttle between PQQ-GDH containing E. coli cells and electrode [3]. Obviously, the electron exchange reaction between enzyme-bound PQQ cofactor and soluble NMP proceeds at a high rate, enabling an efficient bioelectrocatalytic reaction to proceed.…”

Measurements of Reversible and Irreversible Inactivation Processes of a Redox Enzyme, Bilirubin Oxidase, by Electrochemical Methods Based on Bioelectrocatalysis

“…7 The use of immobilized-biocatalyst electrodes has been reported for studies of the activation and deactivation of biocatalysts. 8,9 The method is expected to provide a means for evaluating the stability of an enzyme under immobilized conditions. This is a method appropriate for enzymes designed for industrial use, because they are often used in their immobilized states.…”