ABSENCE OF OBSERVABLE BIOTIN-PROTEIN INTERACTIONS IN THE 1.3S SUBUNITOF TRANSCARBOXYLASE - AN NMR-STUDY

Transcarboxylase (TC) is a biotin-containing enzyme catalyzing the tra nsfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form p ropionyl-CoA and oxalacetate. The transfer is achieved via carboxylate d biotin bound to a 1.3S subunit within the multisubunit enzyme comple x. The 1.3S subunit of TC is a 123 amino acid polypeptide. to which bi otin is covalently attached at Lys 89. We have overexpressed 1.3S in E scherichia coli and characterized the biotinylated and ape-forms by 1D - and 2D-NMR spectroscopy. To search for protein-biotin interactions, which could modulate the reactivity of the biotin ring on the 1.3S sub unit, Mie have compared the chemical shifts, relaxation parameters, an d NH exchange rates of the ureido ring protons of free and 1.3S-bound biotin. These properties are similar for both forms of the biotin, Fur ther, NOE experiments on 1.3S revealed no detectable cross peaks betwe en biotin and the protein. Consistent with these findings, the 2D NMR data for hole-and apo-1.3S are essentially identical indicating little or no changes in conformation between the two forms of the protein. T he conclusion that strong protein-biotin interactions do not exist in 1.3S contrasts with the findings for the biotin carboxylase carrier pr otein from E. coli acetyl-CoA carboxylase, which reveal significant bi otin-protein contacts [Athappilly, F. K., and Hendrickson, W. A. (1995 ) Structure 3, 1407-1419]. Further, the biotin NH1' exchange rates det ermined fur 1.3S show that in the region of optimal activity or TC (pH 5.5-6.5) acid-catalyzed exchange predominates. Tn this pH range the b ase-catalyzed rate is too small (<1 s(-1)) to account for the turnover rate of the enzyme. Thus, the means by which the N1' atom is activate d for nucleophilic attack of the carboxyl group in methylmalonyl-CoA d oes not appear to depend on interactions within the 1.3S subunit alone ; rather activation must occur at the interfaces of the subunits in th e holoenzyme.