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.
KeywordsPseudomonas Fluorescens Glucose Dehydrogenase Acinetobacter Calcoaceticus Active Site Model Pyrroloquinoline Quinone
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