YEAST ALPHA-ISOPROPYLMALATE SYNTHASE: COENZYME A INACTIVATION, LEUCINE INHIBITION AND SUBCELLULAR LOCATION
Abstract
Yeast (alpha)-isopropylmalate ((alpha)-IPM) synthase (E.C. 4.1.3.12) is inactivated by micromolar concentrations of CoA in the presence of Zn('2+). It is reported here that rapid reactivation of inactivated enzyme occurred in the presence of millimolar concentrations of ATP or ADP, using permeabilized cells. For the reactivation of purified, CoA-zinc inactivated enzyme, a chelator was required in addition to ATP. Reactivation was also possible by processes that remove CoA from equilibrium; however, these processes were slow. Conditions are defined that result in protection against CoA-zinc inactivation. Since other nucleoside triphosphates are less effective than ATP it is concluded that the ATP effect is a specific adenylate effect. Studies on the mechanism of CoA inactivation showed that inactivation does not involve covalent modification, but is more likely the result of the formation of an enzyme(.)CoA(.)zinc complex held together by noncovalent forces. Two 5',5',5'-trifluoroleucine-resistant mutants of Saccharomyces cerevisiae S288c(alpha) containing feedback resistant (alpha)-IPM synthase were found to overproduce and excrete leucine; excretion roughly paralleled the degree of feedback resistance. In the feedback-resistant mutants, the levels of all leucine pathway-specific enzymes were elevated, and the repressive effect on IPM isomerase and (beta)-IPM dehydrogenase by leucine plus theronine had largely disappeared. An isomerase-negative, feedback-resistant double mutant excreted (alpha)-IPM and exhibited a very high dehydrogenase level. These results are discussed in terms of a possible regulatory mechanism for isomerase and dehydrogenase expression. (alpha)-IPM synthase was previously shown to be mitochondrially-associated. Using isolated, intact mitochondria it is reported here that the enzyme was unaffected by externally-added proteases and inaccessible to its substrates. Subfractionation of the mitochondria showed that the enzyme remained with the mitoplasts. Thus, (alpha)-IPM synthase is not just mitochondrially-associated, but is located in the matrix. Cell free synthesis of (alpha)-IPM synthase showed that this enzyme is not synthesized as a larger molecular weight precursor.
Degree
Ph.D.
Subject Area
Biochemistry
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