EVIDENCE FOR SUBSTRATE STABILIZATION IN REGULATION OF THE DEGRADATIONOF BACILLUS-SUBTILIS ASPARTATE-TRANSCARBAMYLASE IN-VIVO

Aspartate transcarbamylase (ATCase) is rapidly degraded in Bacillus su btilis cells that are starved for a carbon or nitrogen source or a req uired amino acid, The hypothesis that ATCase degradation may be regula ted in vivo by protection of the enzyme by substrate binding was teste d by studies of a mutant ATCase (Arg99 to Ala, R99A), which binds subs trate 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 rapid ly in exponentially growing cells. Degradation of the mutant enzyme wa s twofold slower in a relA strain, as was degradation of the normal AT Case, 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 wer e degraded identically in vitro by subtilisin, except that the mutant enzyme was much less effectively protected against cleavage by carbamy l phosphate, as expected, The carbamyl phosphate pool in glucose-limit ed B. subtilis cells was only one-third of the pool in exponentially g rowing cells. These results indicate that protection of ATCase by carb amyl phosphate binding could be one of the elements that regulate ATCa se stability in vivo, However, carbamyl phosphate pools were the same in cells grown with ammonium ions,and with a mixture of 20 common amin o acids, conditions under which ATCase stability in vivo differs, Thus , other means of regulating ATCase degradation must also exist. (C) 19 95 Academic Press, Inc.