Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase.
Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).
Elias
M D
MD
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
Tanaka
M
M
Izu
H
H
Matsushita
K
K
Adachi
O
O
Yamada
M
M
D001665:Binding Sites; D002384:Catalysis; D003067:Coenzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000737:chemistry* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D045542:PQQ Cofactor; D015363:Quinolones / Q000737:chemistry*; D011809:Quinones / Q000737:chemistry*; D013329:Structure-Activity Relationship
D003067:Coenzymes; D015363:Quinolones; D011809:Quinones; D045542:PQQ Cofactor; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D005947:Glucose