136 HILLEL S. LEVINSON 



The amidation of glutamic acid to glutamine through glutamine synthetase 

 occurs in spore homogenates, spore-free extracts, and vegetative cells of 

 B. cereus. The enzyme was studied by substituting h\droxylamine for NH3 

 and determining glutamine as glutamohydroxamic acid (GHA). The spore 

 enzyme has a pH optimum of 7.2 and requires Mg++ and ATP. Mn+ + 

 could not be tested satisfactorily since precipitates form at low Mn++ con- 

 centrations in homogenate preparations. Mn++ does, however, satisfy the 

 metal ion requirement of GS from other bacterial sources ( Fry, 1954) . 



This enzyme is of particular interest because early studies in our labora- 

 tory on the sporulation of B. subtilis in a simple glucose-glutamic acid-salts 

 medium led to the postulate that this reaction was involved in sporulation 

 (Krask, 1953). Sporulation of B. subtilis was dependent on the glutamic 

 acid concentration. The inclusion of methionine sulfoxide, a known inhibitor 

 of the conversion of glutamic acid to glutamine, resulted in an inhibition of 

 sporulation without affecting growth. These results were the stimulus for 

 an investigation of enzymes metabolizing glutamic acid. 



(2) Glutamotransf erase (GTF) 



Mn, ATP 

 Glutamine -|- NH^OH ^ Glutamohydroxamic acid (GHA) 



The verification of the synthesase reaction in spores and cells indicated that 

 glutamine might be related to spore formation. Of the many reactions in- 

 volving glutamine, one in particular — catalysis by glutamotransferase — ap- 

 peared attractive in terms of a relationship to synthetic processes. Glutamo- 

 transferase catalyzes the enzymatic exchange of the amide group of glutamine 

 with hydroxylamine to form glutamohydroxamic acid (Waelsch et al, 1950; 

 Grossowicz et al, 1950). The reaction may be considered as the transfer 

 of the gamma-glutamyl radical from glutamine to an acceptor hydroxylamine. 

 Waelsch and his coworkers ascribe particular importance to this reaction in 

 peptide synthesis. They suggest that the reaction of glutamine with hy- 

 droxylamine is only a model for a biologically significant reaction in which 

 the gamma-glutamyl radical combines with amino acids to form gamma- 

 glutamyl peptides. 



This enzyme occurs in spore homogenates and extracts and in the vegeta- 

 tive cells. The spore enzyme has a pH optimum of 7.4-7.45 and is almost com- 

 pletely inactive at pH 6.0 and 9.0. In contrast to GTF from other sources, 

 spore GTF is satisfied by Mn + + and ATP only. Mg++ and Co++ are 

 inactive as is ADP and AMP. It is of interest that Williams and Thome 

 (19.54) and Williams et al (1955) found a type of GTF in exoenzyme prepa- 

 rations of B. subtilis cells which catalyzed transamidation reactions between 

 glutamine and amino acids and transpeptidation reactions between gamma- 

 glutamylglutamic acid to form gamma-glutamyl peptides of greater chain 



