Absence of observable biotin-protein interactions in the 1.3S subunit of transcarboxylase: an NMR study.

ABSTRACT

Transcarboxylase (TC) is a biotin-containing enzyme catalyzing the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxalacetate. The transfer is achieved via carboxylated biotin bound to a 1.3S subunit within the multisubunit enzyme complex. The 1.3S subunit of TC is a 123 amino acid polypeptide, to which biotin is covalently attached at Lys 89. We have overexpressed 1.3S in Escherichia coli and characterized the biotinylated and apo-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 subunit, we have compared the chemical shifts, relaxation parameters, and 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. Further, NOE experiments on 1.3S revealed no detectable cross peaks between biotin and the protein. Consistent with these findings, the 2D NMR data for holo- and apo-1.3S are essentially identical indicating little or no changes in conformation between the two forms of the protein. The conclusion that strong protein-biotin interactions do not exist in 1.3S contrasts with the findings for the biotin carboxylase carrier protein from E. coli acetyl-CoA carboxylase, which reveal significant biotin-protein contacts [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419]. Further, the biotin NH1' exchange rates determined for 1.3S show that in the region of optimal activity for TC (pH 5.5-6.5) acid-catalyzed exchange predominates. In this pH range the base-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 activated for nucleophilic attack of the carboxyl group in methylmalonyl-CoA does not appear to depend on interactions within the 1.3S subunit alone; rather activation must occur at the interfaces of the subunits in the holoenzyme.