The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Characterization of Bacillus subtilis Glutamine Synthetase by Limited Proteolysis
Kinuko KimuraShigeyoshi SuganoAkihiro FunaeYoshio Nakano
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JOURNAL FREE ACCESS

1991 Volume 110 Issue 4 Pages 526-531

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Abstract

The inactivation of native glutamine synthetase (GS) from Bacillus subtilis by trypsin, chymotrypsin, or subtilisin followed pseudo-fast order kinetics. Trypsin cleaved the polypeptide chain of GS into two principal fragments, one of about 43, 000 (Mr) and the other of smaller than 10, 000. Chymotrypsin and subtilisin caused similar cleavage of GS. A large fragment (Mr 35, 000) and one smaller than 10, 000 were detected on SDS-PAGE. The nicked protein remained dodecameric, as observed on gel filtration, electrophoresis, and electron micrography. In the presence of glutamate, ATP, and Mn2+, the digestion of GS by each of the three proteases was retarded completely; however, the presence of one substrate, L-glutamate, ATP+Mn2+, or ATP+Mg2+ led to partial protection. The product, L-glutamine, did not retard but altered the susceptibility of the protease sensitive sites. Amino acid sequence analysis of the two smaller polypeptide fragments showed that the nicked region was around serine 375 and serine 311, respectively, and that both large fragments (43, 000 and 35, 000) were N-terminal polypeptides of GS. The serine 311 region was involved in the formation of the enzyme-substrate complex. Tyrosine 372 near serine 375 corresponded to tyrosine 397 which was adenylylated by adenyltransferase in Escherichia coli GS.

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© The Japanese Biochemical Society
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