scholarly journals Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation

1989 ◽  
Vol 261 (2) ◽  
pp. 415-421 ◽  
Author(s):  
O Geiger ◽  
H Görisch
Keyword(s):  
Acetic Acid ◽  
Active Site ◽  
Protein Concentration ◽  
Thermal Inactivation ◽  
Glucose Dehydrogenase ◽  
Acinetobacter Calcoaceticus ◽  
Pyrroloquinoline Quinone ◽  
Soluble Form ◽  
Activation Process ◽  
Prosthetic Group

The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture.

Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone

2001 ◽  
Vol 357 (3) ◽  
pp. 893-898 ◽  
Author(s):  
Akihiro SATO ◽  
Kazuyoshi TAKAGI ◽  
Kenji KANO ◽  
Nobuo KATO ◽  
Johannis A. DUINE ◽  
...  
Keyword(s):  
Redox Potential ◽  
Active Site ◽  
Continuous Flow ◽  
Glucose Dehydrogenase ◽  
Acinetobacter Calcoaceticus ◽  
Substrate Oxidation ◽  
Pyrroloquinoline Quinone ◽  
Free State ◽  
Semiquinone Radical ◽  
Chemical Mechanisms

Spectroelectrochemical studies were performed on the interaction between Ca2+ and pyrroloquinoline quinone (PQQ) in soluble glucose dehydrogenase (sGDH) and in the free state by applying a mediated continuous-flow column electrolytic spectroelectrochemical technique. The enzyme forms used were holo-sGDH (the holo-form of sGDH from Acinetobacter calcoaceticus) and an incompletely reconstituted form of this, holo-X, in which the PQQ-activating Ca2+ is lacking. The spectroelectrochemical and ESR data clearly demonstrated the generation of the semiquinone radical of PQQ in holo-sGDH and in the free state in the presence of Ca2+. In contrast, in the absence of Ca2+ no semiquinone was observed, either for PQQ in the free state (at pH7.0) or in the enzyme (holo-X). Incorporation of Ca2+ into the active site of holo-X, yielding holo-sGDH, caused not only stabilization of the semiquinone form of PQQ but also a negative shift (of 26.5mV) of the two-electron redox potential, indicating that the effect of Ca2+ is stronger on the oxidized than on the reduced PQQ. Combining these data with the observations on the kinetic and chemical mechanisms, it was concluded that the strong stimulating effect of Ca2+ on the activity of sGDH can be attributed to facilitation of certain kinetic steps, and not to improvement of the thermodynamics of substrate oxidation. The consequences of this conclusion are discussed for the oxidative as well as for the reductive part of the reaction of sGDH.

The 9-carboxyl group of pyrroloquinoline quinone, a novel prosthetic group, is essential in the formation of holoenzyme of D-glucose dehydrogenase

1986 ◽  
Vol 139 (3) ◽  
pp. 1279-1284 ◽  
Author(s):  
Emiko Shinagawa ◽  
Kazunobu Matsushita ◽  
Masatsugu Nonobe ◽  
Osao Adachi ◽  
Minoru Ameyama ◽  
...  
Keyword(s):  
Glucose Dehydrogenase ◽  
Pyrroloquinoline Quinone ◽  
Carboxyl Group ◽  
Prosthetic Group

scholarly journals Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of methanol dehydrogenase from Methylobacterium extorquens

1995 ◽  
Vol 312 (3) ◽  
pp. 679-685 ◽  
Author(s):  
G E Cozier ◽  
C Anthony
Keyword(s):  
Active Site ◽  
Glucose Dehydrogenase ◽  
Ring Structure ◽  
Enzymic Activity ◽  
Methanol Dehydrogenase ◽  
The Novel ◽  
Methylobacterium Extorquens ◽  
Prosthetic Group ◽  
Site Structure ◽  
Proposed Model

The structure of methanol dehydrogenase (MDH) at 0.194 nm (1.94 A) has been used to provide a model structure for part of a membrane quinoprotein glucose dehydrogenase (GDH). The basic superbarrel structure is retained, along with the tryptophan-docking motifs. The active-site regions are similar, but there are important differences, the most important being that GDH lacks the novel disulphide ring structure formed from adjacent cysteines in MDH; in GDH the equivalent region is occupied by His-262. Because of the overall similarities in the active-site region, the mechanism of action of GDH is likely to be similar to that of MDH. The differences in co-ordination to the cation and bonding to the pyrrolo-quinoline quinone (PQQ) in the active site may explain the relative ease of dissociation of the prosthetic group from the holo-GDH. There are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site, the configuration of which influences substrate specificity. The proposed model is consistent in many respects with previous proposals for the active-site structure based on the effects of chemical modification on binding of PQQ and enzymic activity.

Prosthetic Group of Aldehyde Dehydrogenase in Acetic Acid Bacteria Not Pyrroloquinoline Quinone

1994 ◽  
Vol 58 (11) ◽  
pp. 2082-2083 ◽  
Author(s):  
Hiroshi Takemura ◽  
Takayasu Tsuchida ◽  
Fumihiro Yoshinaga ◽  
Kazunobu Matsushita ◽  
Osao Adachi
Keyword(s):  
Acetic Acid ◽  
Aldehyde Dehydrogenase ◽  
Acetic Acid Bacteria ◽  
Pyrroloquinoline Quinone ◽  
Prosthetic Group

scholarly journals Pyrroloquinoline Quinone-Dependent Dehydrogenases from Ketogulonicigenium vulgare Catalyze the Direct Conversion of l-Sorbosone to l-Ascorbic Acid

2006 ◽  
Vol 72 (2) ◽  
pp. 1487-1495 ◽  
Author(s):  
Taro Miyazaki ◽  
Teruhide Sugisawa ◽  
Tatsuo Hoshino
Keyword(s):  
Ascorbic Acid ◽  
Glucose Dehydrogenase ◽  
Acinetobacter Calcoaceticus ◽  
Direct Conversion ◽  
Pyrroloquinoline Quinone ◽  
Binding Motif ◽  
Bacterial Cells ◽  
Ketogulonicigenium Vulgare ◽  
Gene Coding ◽  
Prosthetic Groups

ABSTRACT A novel enzyme, l-sorbosone dehydrogenase 1 (SNDH1), which directly converts l-sorbosone to l-ascorbic acid (l-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced l-AA, as well as 2-keto-l-gulonic acid (2KGA), from l-sorbosone, suggesting that tautomerization of l-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.

scholarly journals Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine

1999 ◽  
Vol 340 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Gyles E. COZIER ◽  
Raff A. SALLEH ◽  
Christopher ANTHONY
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Active Site ◽  
Marked Decrease ◽  
Glucose Dehydrogenase ◽  
Catalytic Efficiency ◽  
Prosthetic Group ◽  
Competitive Inhibitors ◽  
A Site

The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH 7 and pH 8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process.

scholarly journals Thermodynamics and stoicheiometry of the binding of substrate analogues to uricase

1980 ◽  
Vol 187 (3) ◽  
pp. 727-732 ◽  
Author(s):  
T G Conley ◽  
D G Priest
Keyword(s):  
Metal Content ◽  
Protein Concentration ◽  
Thermal Inactivation ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Enthalpy Change ◽  
Prosthetic Group ◽  
Subunit Dissociation ◽  
Substrate Analogues ◽  
Thermal Stability Of

The subunit composition, metal content, substrate-analogue binding and thermal stability of Aspergillus flavus uricase were determined. A. flavus uricase is a tetramer and contains no copper, iron or any other common prosthetic group. Analytical-gel-filtration and equilibrium-dialysis experiments showed one binding site per subunit for urate analogues. The free energy of xanthine binding was −30.5 kJ (-7.3 kcal)/mol of subunit by equilibrium dialysis and −30.1 kJ (-7.2 kcal)/mol of subunit by microcalorimetry. The enthalpy change for xanthine binding was −15.9 kJ (-3.8 kcal)/mol of subunit when determined from the temperature-dependence of the equilibrium constant and −18.0 kJ (-4.3 kcal)/mol of subunit when measured microcalorimetrically. The thermal inactivation rate of A. flavus uricase increases as protein concentration is decreased. This concentration-dependent instability is not due to subunit dissociation.

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