Sulfhydryl Group Reactivity of d‐Galactose Dehydrogenase from Pseudomonas saccharophila

Abstract

1. Native D-galaCtOSe dehydrogenase from Pseudomonas saccharophila contains 12 SH-groups and no disulfide bonds. Storage of enzyme causes a decrease of enzymatic activity, a loss of SHgroups, and the formation of disulfide bonds. An approximately linear relationship exists between specific activity and the number of SH-groups per mole enzyme for eight of the 12 SH-groups. 2. On the basis of their reactivity against 5,5'-dithio-bis(Z-nitrobenzoic acid), the SH-groups of the enzyme can be differentiated in three classes, each containing four SH-groups.3. The catalytic activity is completely lost upon stoichiometric modification of the first four SH-groups, which react readily with 5,5'-dithio-bis(2-nitrobenzoic acid). The inactivation follows pseudo-first-order kinetics with a second-order rate constant of k = 1.3 mM-l s-l, and is completely reversible upon the addition of reducing agents, such as 2-mercaptoethanol or 1,4-dithioerythritol.4. Evidence is presented that the modification of these first four SH-groups does not occur with the enzyme NAD(H) complex; only the free enzyme is subject to the reaction with Nbs,. The presence of D-galactose or L-arabinose has no influence on the rate of inactivation.5. I n contrast to the SH-groups of the second and third class, the first four are generally not involved in intra-and intermolecular disulfide bridges of the enzyme. galactose dehydrogenase from Pseudomonas saccharophila, an NAD-dependent enzyme with a molecular weight of 102000, is composed of four polypeptide chains each of a molecular weight of 25000. The enzyme contains 12 cysteinyl residues per mole of enzyme, in accordance with the total number of free sulfhydryl groups, and it is probable that therc are three sulfhydryl groups per polypeptide chain [l].By stereoselective inhibition of D-galactose dehydrogenase with the antipodes of 2-iodopropionamide it has been indicated that a sulfhydryl group is located in the area of the active site [ 2 ] .A decrease in the specific activity and the formation of oligomeric artefact forms of the native enzyme is observed as a result of storage in solution [3]. Both Abbreviations. Nbs,, 5,5'-dithio-bis(2-nitrobenzoic acid) ; Nbs-, 1 -carboxy -2 -nitrophenyl -5 -thio-; SH-group, sulfhydryl group.

Enzymatic rearrangement of flavanone to isoflavone

“…In each case the radioactive product coincided exactly with genistein. Furthermore, the product purified by HPLC was methylat~ in presence of S-adoMet with an enzyme from Cicer arietinum seedlings [12]. The with an authentic reference substance.…”

d‐Glucose‐6‐Phosphate Dehydrogenase (Entner‐Doudoroff Enzyme) from Pseudomonas fluorescens

“…The formation of artefact forms by oxidation of sulfhydryl groups (as described for D-galactose dehydrogenase from Ps. saccharophila [35,36] and also known for ~-glucose-6-phosphate dehydrogenase from rat liver [37,38]) as well as the possibility that a change of activity, substrate and coenzyme specificity due to oxidation (as reported for human ~-glucose-6-phosphate dehydrogenase [39]) had occurred, may be completely ruled out because all procedures were performed in the presence of 0.1 M 2-mercaptoethanol. However, other interconversions among multiple forms, which may have a regulatory significance could not be excluded at this stage.…”