Wiring of PQQ-dehydrogenases.
The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.
Laurinavicius
Valdas
V
Institute of Biochemistry, Mokslininku 12, 2600 Vilnius, Lithuania. valdasl@bchi.lt
Razumiene
Julija
J
Ramanavicius
Arunas
A
Ryabov
Alexander D
AD
D000327:Adsorption; D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry; D005947:Glucose / Q000032:analysis* / Q000737:chemistry*; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry* / Q000145:classification; D006863:Hydrogen-Ion Concentration; D008422:Materials Testing; D045542:PQQ Cofactor / Q000737:chemistry*; D011485:Protein Binding; D013379:Substrate Specificity
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D045542:PQQ Cofactor; D000426:Alcohol Dehydrogenase; D042843:Glucose 1-Dehydrogenase; D005947:Glucose