ROLES OF AMINO-ACID-RESIDUES SURROUNDING PHOSPHORYLATION SITE-1 OF BRANCHED-CHAIN ALPHA-KETOACID DEHYDROGENASE (BCKDH) IN CATALYSIS AND PHOSPHORYLATION SITE RECOGNITION BY BCKDH KINASE

Branched-chain alpha-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1 alpha subunit. Alani ne-scanning mutagenesis was used to examine the roles of residues surr ounding serine 293 in catalysis by the dehydrogenase and in sub strate recognition by branched-chain alpha-ketoacid dehydrogenase kinase. Al anine substitution of serine 293 resulted in a 10-fold increased K-m f or alpha-ketoisovalerate, a less increased (2.8-fold) K-m for alpha-ke toisocaproate, but no change in V-max or the K-m for thiamine pyrophos phate. Alanine substitutions of arginine 288, histidine 292, and aspar tate 296, residues highly conserved among alpha-ketoacid dehydrogenase s, 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 alpha-ketoacid dehy drogenase kinase with similar rates, with the exception of the R288A m utant, which displayed no detectable phosphorylation. Thiamine pyropho sphate inhibited the phosphorylation of all mutant enzymes with the ex ception of H292A, the mutant E1 that did not bind thiamine pyrophospha te.