Evidence for an essential histidine residue in glucose dehydrogenase from Bacillus megaterium and sequence analysis of the peptides labeled with bromoacetyl pyridine

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Abstract
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. The inactivation is correlated with the modification of a single histidine residue. Modification of the enzyme with 3‐(2‐bromo[carbonyl‐14C]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‐His‐Glu‐Trp‐Lys‐Ile‐Pro‐Trp‐Pro‐. 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%.