The recently determined primary structure of glucose dehydrogenase from ~a~i~~as megater~~m was scanned by computerized comparisons for similarities with known polyol and alcohol dehydrogenases. The results revealed a highly significant similarity between this glucose dehydrogenase and ribitol dehydrogenase from Klebsiella aerogenes. Sixty-one positions of the 262 in glucose dehydrogenase are identical between these two proteins (23% identity), fitting into a homology alignment for the complete polypeptide chains. The extent of similarity is equivalent to that between other highly divergent but clearly related dehydrogenases (two zinc-containing alcohol dehydrogenases, 25%; sorbitol and zinc-containing alcohol dehydrogenases, 25%; ribitol and non-zinc-containing alcohol dehydrogenases, 20%), and suggests an ancestral relationship between glucose and ribitol dehydrogenases from different bactera. The similarities fit into a previously suggested evolutionary scheme comprjsing short and long aIcohol and polyol dehydrogenases, and greatly extend the former group to one composed of non-zinc-containing alcoholpolyol-glucose dehydrogenases.

The amino acid sequence of glucose dehydrogenase from Bacillus megaterium has been determined. The enzyme consists of 4 identical subunits, each containing 262 amino acid residues. Its structure was established using manual Edman degradation procedures after modification of the enzyme in the native form with reagents specific to the amino acids histidine, tyrosine, tryptophan and lysine in order to identify residues involved in catalysis or located in the subunit binding area. Glucose dehydrogenase Dehydrogenase Modification Bacillus megateriumAmino acid sequence

1. The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residuejsubunit. The nitration does not influence the dissociation-reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP.2. The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr-254 in the covalent structure of the enzyme.3. After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer.4. Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr-254 which is essential for the catalytic activity and the other one is Tyr-160 which seems to be located in the subunit binding area.Information about the precise structures of enzymes, including the active site, is only available when their complete amino acid sequence and three-dimensional structure are known. However, modifications of amino acid side chains with specific reagents can provide information about amino acid residues which are involved in catalysis or ligand binding.Glucose dehydrogenase from Bacillus megaterium catalyses the oxidation of /3-D-glucose to ~-glucono-l,5-lactone using NAD or NADP as coenzyme. The enzyme is a tetrameric protein consisting of subunits identical in size (Mr 30000) and charge [l]. The enzyme shows the unusual ability to dissociate into its inactive monomers at pH 9 and their reassociation, combined with a complete reactivation, at pH 6.5 [2].Following our studies on the structural features and the dissociation-reassociation process of glucose dehydrogenase [3 -51, we now report the effects upon modification of the enzyme in the tetrameric and monomeric states with tetranitromethane and the identification of the modified tyrosine residues. MATERIALS AND METHODS ChemicalsAnalytical grade reagents, primarily from E. Merck (Darmstadt), were used. NAD, AMP, ATP, ADP, adenosineAbbreviations. BNPS-skatole, 2-(2-nitrophenylsulfenyl)-3-bromoindolenine; DABTH, dimethylaminoazobenzenthiohydantoin; dansyl, 5-dimethylaminonaphthalene-I -sulfonyl; N02-Tyr, nitrotyrosyl ; TLC, thin-layer chromatography; HPTLC, high-pressure thin-layer chromatography. Peptides generated by cleavage with CNBr are designated with the prefix, CN ; with BNPS-skatole, BS ; with trypsin, TA or t; with chymotrypsin, c; with Staphylococcus aweus V8 protease, st; with thermolysin, th.Enzymes. Glucose dehydrogenase, P-1)-glucose :NAD(P) and adenine were obtained from Boehringer (Mannheim) ; ADP-ribose and NMN were purchased from Sigma (Munich). Tetranitromethane, cyanogen bromide and BNPS-skatole were purchased from Serva (Heidelberg). Dimethylaminoazobenzene-4-isothiocyanate was obtained from Fluka and recrystallized [6]. Butylacetate and pyridine were redistilled ...

1. Bromoacetylpyridine acts as an active-site-directed inhibitor on glucose dehydrogenase from Bacillus megaterium. The inactivation is irreversible with a Ki of 7.7 mM. The coenzyme NAD but not the substrate glucose protects the enzyme from the inactivation. It is proposed that bromoacetylpyridine modifies a residue at or nearby the active site.2. The inactivation is correlated with the modification of a single histidine residue.3. Modification of the enzyme with 3-(2-bromo[c~rbunyl-'~C]acetyl)-pyridine and partial acid hydrolysis of the protein yielded one labeled fragment. From the arginine restricted tryptic cleavage of this fragment four radioactively labeled peptides were purified. Comparison of the specific radioactivity leads to the conclusion that the active site histidine residue must be located in the 58-residue fragment AH2-TA3. Sequence analysis showed that only one residue is modified in this fragment and the sequence around the labeled histidine residue is -Met-Ser-Ser-Val-HisGlu-Trp-Lys-Ile-Pro-Trp-Pro-.4. The minor labeled arginine fragments, comprising 86, 20 and 13 residues, were also sequenced. Only lysine residues are modified in these peptides. The modification of the individual residues does not exceed 10 %.Glucose dehydrogenase from Bacillus megaterium catalyses the oxidation of fl-D-glucose to D-gIUCOn0-1 ,5-lactone using NAD or NADP as coenzyme. The enzyme is a tetrameric protein (Mr 118000) consisting of subunits identical in size and charge [I]. The enzyme shows the unusual ability of a dissociation into its inactive monomers at pH 9 and their reassociation combined with a complete reactivation at pH 6.5 [2].An understanding of molecular processes involved in catalysis and dissociation-association requires the knowledge of the covalent structure of the enzyme and especially of the functional groups participating in binding of ligands and interactions between the subunits. Chemical modifications of the enzyme in the tetrameric or monomeric state with specific reagents followed by enzymatic and physico-chemical methods and sequence analysis were done as an approach to identify such residues. Glucose dehydrogenase has some advantages for such an investigation. Unlike many other dehydrogenases, glucose dehydrogenase contains no cysteine residues [l]. Thiol groups react with many modification reagents and their undesired modification can lead to a misinterpretation of the results. The enzyme is very stable and at high ionic strength or in the presence of NAD the dissociation can be prevented even at pH 9. The half-life time of the monomers is several hours. A systematic investigation is being carried out on glucose dehydrogenase in our laboratory. In the present paper we report the effect of bromoacetylpyridine on glucose dehydrogenase and the sequence around the modified histidine residue which is probably part of the active center of the enzyme. MATERIALS A N D METHODS ChemicalsAnalytical grade reagents, primarily from Merck (Darmstadt) were used. 3-(2-brorno[c~rbonyl-~~C]acetyI)pyridine...