Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains

Biotinylation in vivo is an extremely selective posttranslational event whe re the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met m otif, resides in a structured domain that functions as the BPL substrate. W e have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated u sing this strategy were analyzed by in vivo biotinylation assays performed at both 30 degreesC and 37 degreesC, The temperature-sensitive substrates w ere reasoned to have structural mutations, leading to compromised conformat ions at the higher temperature. This interpretation was supplemented by mol ecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr p roduced mutations that were temperature-insensitive in the in vivo assay. F urthermore, these two mutant proteins and wild-type yPC-104 showed identica l susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purifi ed Met --> Thr/Val mutant proteins with both yeast and Escherichia coil BPL s revealed that these substitutions had a strong effect upon K-m values but not k(cat). The Met --> Thr mutant was a poor substrate for both BPLs, whe reas the Met --> Val substitution was a poor substrate for bacterial BPL bu t had only at S-fold lower affinity for yeast BPL than the wild-type peptid e. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-lys results in mutants specifically compromised in their in teraction with BPL.