The conserved methionines of the 1.3 S biotinyl subunit of transcarboxylase: effect of mutations on conformation and activity.

Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. Transcarboxylase 26 S complexes consist of a central, hexameric 12 S subunit with 6 outer, 5 S subunits attached by 12 1.3 S biotinyl subunits. Each of the subunits has been cloned and expressed in Escherichia coli in active form. We have used the cloned genes in mutagenic studies of the structure-function interactions of these subunits. One particular target of our studies has been the evolutionarily conserved tetrapeptide Ala-Met-Bct-Met which surrounds the biotinyl lysine. We have investigated the properties of subunits containing leucine substitutions at each methionine (1.3 S M88L and 1.3 S M90L) by assaying their activity in the two partial reactions in which this subunit participates. Partial reaction assays demonstrate that leucine substitution at either position has a greater effect on the 12 S partial reaction than on the 5 S reaction and Met 88 is more significant catalytically than Met 90. To determine whether structural alterations in the 1.3 S mutants were responsible for the effects on activity, the conformations of these mutants were investigated. In vitro hydrolysis studies with trypsin and V8 protease demonstrated differences in the susceptibility of 1.3 S M88L relative to 1.3 S WT and 1.3 S M90L. Complexes of avidin with 1.3 S WT or mutant subunits, as monitored by fluorescence properties, indicated that the microenvironment of the biocytin of 1.3 S M88L was different from those of 1.3 S WT and 1.3 S M90L. By contrast, substrate binding (oxalacetate for 5 S and methylmalonyl-CoA for 12 S) was unaffected by any of the 1.3 S mutants. Taken together, these results indicate that the conserved tetrapeptide of the 1.3 S biotinyl subunit, particularly Met 88, is required to provide an essential conformation and proper binding properties for catalysis of the partial reactions and the overall reaction.