Branched-chain α-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1α subunit. Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched- chain α-ketoacid dehydrogenase kinase. Alanine substitution of serine 293 resulted in a 10-fold increased K(m) for α-ketoisovalerate, a less increased (2.8-fold) K(m) for α-ketoisocaproate, but no change in V(max) or the K(m) for thiamine pyrophosphate. Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among α-ketoacid dehydrogenases, resulted in inactive enzymes. Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1. Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate. Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain α-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation. Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiomine pyrophosphate.