Yujiro Imanaga

Investigations on the Active Site of Glucose Dehydrogenase from Pseudomonas fluorescens

Effects of several inhibitors on the glucose dehydrogenase (EC 1. 1.99.17) from Pseudomonas fluorescens were studied. 2,3-Butanedione (under room light), 8-anilinonaphthalenesulfonate, ethoxyformic anhydride and rosebengal (under irradiation with tungsten lamp) inactivated especially apoenzyme, and Arg and His residue are presumed to participate in the formation of holoenzyme complex. p-chloromercuribenzoate and p-chloromercuri-benzenesulfonate also inactivated apoenzyme, but the inactivation was prevented by preincubation with Ca2+ alone. N-acetylimidazole showed similar effects on apoenzyme, the activity being restored by hydroxylamine-treatment of the inactivated apoenzyme. These results suggest that both Cys and Tyr residues of apoenzyme bind directly to Ca2+ in holoenzyme complex. N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, on the contrary, inactivated especially holoenzyme, and both inactivations were accelerated in the presence of glucose. These results suggest that another type of Cys residue participates directly in the enzyme reaction. Phenylhydrazine inactivated holoenzyme and glucose protected holoenzyme against the inactivation. CN- inhibited holoenzyme reversibly and competed with substrate glucose in the enzyme reaction. These results suggest the interaction of C(5)-carbonyl group of pyrroloquinoline quinone (PQQ) with glucose. Ca2+ promoted the non-enzymic reduction of PQQ by glucose, but was rather inhibitory to the reoxidation process. An active site model and a possible reaction mechanism are proposed.

The synthesis of 6-O-(2-acetamido-2-deoxy-β-D-glucopyranosyluronic acid)-D-glucose

Abstract Two routes for the synthesis of 6- O -(2-acetamido-2-deoxy-β- D -glucopyranosyluronic acid)- D -glucose ( 12 ) were studied. In the first, benzyl 6- O -(2-acetamido-2-deoxy-β- D -glucopyranosyl)-β- D -glucopyranoside ( 10 ) was obtained by condensation of benzyl 2,3,4-tri- O -acetyl-β- D -glucopyranoside (obtained via the 6- O -trityl derivative) with 3,4,6-tri- O -acetyl-2-deoxy-2-diphenylphosphorylamino-α- D -glucopyranosyl bromide, followed by removal of the diphenylphosphoryl and O -acetyl groups and subsequent N -acetylation. In the second route, 2-methyl-4,5-(3,4,6-tri- O -acetyl-2-deoxy-α- D -glucopyrano)-2-oxazoline was used as 2-acetamido-2-deoxy- D -glucose moiety in the condensation reaction to give benzyl 6- O -(2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-β- D -glucopyranosyl)-2,3,4-tri- O -acetyl-β- D -glucopyranoside ( 11 ), and subsequent removal of O -acetyl groups gave compound 10 . Oxidation of the primary hydroxyl group of 10 gave benzyl 6- O -(2-acetamido-2-deoxy-β- D -glucopyranosyluronic acid)-β- D -glucopyranoside ( 8 ), which was converted by catalytic hydrogenolysis into the free disaccharide 12 .

Purification and properties of D-glucosaminate dehydratase from Agrobacterium radiobacter

D-Glucosaminate dehydratase (EC 4.2.1.26)* catalyzes the conversion of D-glucosaminate (GlcNA) to 2-keto3deoxy-D-gluconate and ~monia;it requires pyridox~-S’-phosphate ~yridox~-P) as a cofactor and is analogous to serine dehydratase and threonine dehydratase. The enzyme was discovered independently by Merrick and Roseman [ 1 ] and by Imanaga [2] in the cells of unidentified bacteria in their studies of metabolism of D~luco~m~e. In the former case, the enzyme was formed inducibly by GlcNA, which was used as a sole source of carbon, and partially purified from the cell free extract [3]. It is the exclusive pyridoxal-P enzyme which acts specifically on an amino-sugar derivative. However, since the early works [l-3], little attention has been given to the enzyme. We here describe the purification of the GlcNAdehydrataae from the glucosamineadapted cells of Agrobacterium radiobacter to homogenity, and some of its properties.

Purification and Characterization of d-Glucosaminate Dehydratase from Pseudomonas fluorescens

d-Glucosaminate dehydratase (EC 4.2.1.26) from Pseudomonas fluorescens (IFO 14808) was purified to homogeneity, as judged by the criterion of a single band on disc-gel electrophoresis. The enzyme (molecular weight 61,000) consisted of two subunits identical in molecular weight (about 30,000). Pyridoxal 5′-phosphate was an essential cofactor for the enzyme.The enzyme catalyzed the dehydration of d-glucosaminate and of several d- and l-hydroxyamino acids. Especially when the concentration of substrate was low, d-serine was dehydrated at a rate similar to the rate of dehydration of d-glucosaminate. If both substrates were added to the reaction mixture at the same time, the rate of the reaction was additive until their individual concentrations reached to 0.2 mM level. However, as the concentrations of both the substrates were increased, the rate fell below the rate recorded with d-glucosaminate only. The kinetics of this reaction (in the presence of 1~10mm d-GIcNA) demonstrate that d-glucosaminate dehydratas...

Stereochemistry of an α,β-elimination reaction by D-glucosaminate dehydratase

The proton NMR analysis of D‐glucosaminate dehydratase reaction in D2O revealed the incorporation of a deuterium atom at C‐3 carbon of the product, 2‐keto‐3‐deoxy‐D‐gluconate. Based on the chemical shift of C‐3 proton of the product and the coupling constant characteristic for the C‐3 and C‐4 axial‐axial coupling in the 2C5 pyranose conformation, the deuterium is in the pro‐S position. Thus, the dehydration of D‐glucosaminate by the enzyme proceeds in a retention mode at C‐3 carbon. Kinetic parameters show that the rate‐determining step is the abstraction of α‐proton from the substrate.