Evidence for substrate stabilization in regulation of the degradation of Bacillus subtilis aspartate transcarbamylase in vivo.
Aspartate transcarbamylase (ATCase) is rapidly degraded in Bacillus subtilis cells that are starved for a carbon or nitrogen source or a required amino acid. The hypothesis that ATCase degradation may be regulated in vivo by protection of the enzyme by substrate binding was tested by studies of a mutant ATCase (Arg99 to Ala, R99A), which binds substrate so poorly that it fails to support pyrimidine-independent growth in a pyrB strain, but still has 10% of normal activity when saturated with substrates. Unlike normal ATCase, R99A ATCase was degraded rapidly in exponentially growing cells. Degradation of the mutant enzyme was two-fold slower in a relA strain, as was degradation of the normal ATCase. The stability of purified R99A ATCase to denaturation by heat or guanidine hydrochloride was identical to that of wild-type ATCase, as was its circular dichroic spectrum. The wild-type and R99A ATCase were degraded identically in vitro by subtilisin, except that the mutant enzyme was much less effectively protected against cleavage by carbamyl phosphate, as expected. The carbamyl phosphate pool in glucose-limited B. subtilis cells was only one-third of the pool in exponentially growing cells. These results indicate that protection of ATCase by carbamyl phosphate binding could be one of the elements that regulate ATCase stability in vivo. However, carbamyl phosphate pools were the same in cells grown with ammonium ions and with a mixture of 20 common amino acids, conditions under which ATCase stability in vivo differs. Thus, other means of regulating ATCase degradation must also exist.
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Department of Biochemistry, University of Illinois, Urbana 61801.
Switzer
R L
RL
D001221:Aspartate Carbamoyltransferase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D008025:Ligases / Q000378:metabolism; D009154:Mutation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000378:metabolism; D013381:Subtilisins / Q000494:pharmacology
D006146:Guanidines; D011994:Recombinant Proteins; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; D001221:Aspartate Carbamoyltransferase; D013381:Subtilisins; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases; D019791:Guanidine