| Popis: |
Bacillus subtilis α-amylase is inactivated by treatment with N-acetylimidazole. Full catalytic activity is restored when the acetylated protein is treated with hydroxylamine. Spectral changes associated with the loss and recovery of activity indicate that exposed residues of tyrosine react. If the enzyme is acetylated in the presence of substrate (soluble starch) the modified protein retains 90% of its catalytic activity and one less tyrosyl residue per 24,000 g of protein reacts, compared with the control which has only 20% activity remaining. Free amino groups are acetylated by N-acetylimidazole but hydroxylamine did not remove these acetyl groups, under the conditions used for regeneration of activity. Maleic anhydride reacts with amino groups on the enzyme but there is only limited loss of catalytic activity with extensive maleylation of the protein. Tetranitromethane inactivates B. subtilis α-amylase. The absorption spectrum and amino acid analysis of a sample which had lost 70% of its activity show that 1 to 1.2 residues of tyrosine per 24,000 g of protein are nitrated while no other residues are affected. The apparent pK of the 3-nitrotyrosine in the modified enzyme is 7.7 which is higher than that of the free amino acid derivative (pKapp = 6.8). Peptide maps of a tryptic digest of the modified protein show a single yellow spot. This tryptic peptide contains 1 eq of 3-nitrotyrosine and corresponds with a tyrosine-containing peptide in the map of the native enzyme. These data are interpreted to indicate that in B. subtilis α-amylase tyrosyl residues are required for enzymic activity. Attempts to relate the tyrosyl residue protected by substrate during acetylation with that residue nitrated by tetranitromethane were made, but are not conclusive. The data support the evidence from peptide mapping that in B. subtilis α-amylase the active, physical entity of about 48,000 molecular weight is composed of two equivalent subunits. |
