Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression

Abstract

A selection system has been devised for isolating hexokinase PII structural gene mutants that cause defects in C catabolite repression, but retain normal catalytic activity. Diploid parental strains with homozygotic defects in the hexokinase PI structural gene and with only 1 functional hexokinase PII allele were used. Of 3000 colonies tested, 34 mutants (hex1r) did not repress the synthesis of invertase, maltase, malate dehydrogenase and respiratory enzymes. These mutants had additional hexokinase PII activity. In contrast to hex1 mutants, which were allelic to structural gene mutants of hexokinase PII and had no catalytic activity the hex1r mutants sporulated hardly at all or formed aberrant cells. Those ascospores obtained were mostly inviable. As the few viable hexlr segregants were sterile, triploid cells were constructed to demonstrate allelism between hexlr mutants and hexokinase PII structural gene mutants. Metabolite concentrations, growth rate, and ethanol production were the same in hexlr mutants and their corresponding wild-type strains. Recombination of hexokinase and glucokinase alleles gave strains with different specific activities. The defect in C catabolite repression was strongly associated with the defect in hexokinase PII and was independent of the glucose phosphorylating capacity. Therefore, a secondary effect caused by reduced hexose phosphorylation was not responsible for the repression defect in hex1r mutants. These results, and those with the the hex1r mutants isolated, strongly supported an earlier hypothesis that hexokinase PII is a bifunctional enzyme with catalytic activity and a regulatory component triggering C catabolite repression.