Gluconate Dehydrogenase Membrane Bound

D-Gluconate ^ 2-keto-D-gluconate

D-Gluconate dehydrogenase (GADH) (EC 1.1.99.3) occurs on the outer surface of cytoplasmic membranes of aerobic bacteria, such as Pseudomonas, Klebsiella, Ser-ratia, and acetic acid bacteria. The enzyme activity is linked to the electron transport chain in the cytoplasmic membrane constituting a D-gluconate oxidase system [89-91].

GADH of P. aeruginosa shows a single protein band on native PAGE, but the enzyme preparations from K. Klebsiella and S. marcescens are separated into two protein bands [92]. The low-mobility band is yellow and fluorescent under ultraviolet light, and shows enzyme activity. The coenzyme functioning has been shown to be 8a-[N3-histidyl)riboflavin from the largest subunit [93]. Another protein band with higher mobility is red-colored. In SDS-PAGE, enzyme preparations from four different bacterial strains dissociate into three different polypep-tide bands, subunit I having 66-68 kDa, subunit II (48-52 kDa), and subunit III (22-25 kDa). The sum of the three subunits gives 130-140 kDa as the total molecular mass. GADH is a monomeric protein dispersed in the presence of 0.1% detergent such as Triton X-100, whereas the deletion of detergent allows GADH to be the dimeric enzyme. The removal of detergent from the purified enzyme causes a decrease of enzyme activity. Activation of GADH is observed with phospholipids, especially cardiolipin, in the presence of Triton X-100. Thus, GADH has a hydrophobic and phospholipid-interacting domain, and hence is a typical membrane-bound dehydrogenase.

All GADHs are highly specific for D-gluconate oxidation with Km values of 0.3-0.8 mmol L-1 and show optimum pH at 4.0-5.0, when assayed with potassium ferricyanide. The following compounds are not oxidized by GADH: aldohexoses, aldopentoses, 2-keto-D-gluconate, 5-keto-D-gluconate, D-galactonate, D-man-nonate, 6-phospho-D-gluconate, L-idonate, D-arabonate, and D-xylonate. Since the enzyme activity of GADH is not affected at all by the presence of high concentrations of the above compounds, GADH is a favorable enzyme for D-gluconate measurement [94]. D-Gluconate is well known as one of the natural ingredients of brewing products and is widespread in other materials as food additives. In spite of the wide distribution and usefulness of D-gluconate, an accurate method for its quantitative measurement had not been developed until GADH was proposed for the purpose. A well-known coupling enzymatic D-gluconate determination developed and used so far contains D-gluconate kinase and 6-phospho-D-gluconate dehydrogenase. However, this assay method depends largely on the purity of the enzymes used and is rather expensive. Difficulty in preparing a pure D-gluconate kinase and 6-phospho-D-gluconate dehydrogenase without any contaminants makes this assay system complicated and troublesome. A simple chemical colorimetric determination using GADH from aerobic bacteria is highly reliable method because GADH is prepared with ease and the enzyme activity is stable without appreciable loss if GADH is stored in an appropriate buffer solutions in the presence of detergent. Rate assay as well as the end point measurement for D-gluconate can be done successfully with GADH.

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