SITE-DIRECTED MUTAGENESIS OF PHOSPHORYLATION SITES OF THE BRANCHED-CHAIN ALPHA-KETOACID DEHYDROGENASE COMPLEX

Regulation of the branched chain alpha-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, inv olves phosphorylation of 2 amino acid residues (site 1, serine 293; si te 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutam ates and/or alanines. Functional E1 heterotetramers were expressed in Escherichia coli carrying genes for E1 alpha and E1 beta under control of separate T7 promoters in a dicistronic vector. Mutation of phospho rylation site 1 serine to glutamate inactivated E1 activity, i.e. mimi cked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased K-m for the alpha-ketoacid sub strate but had no effect on maximum velocity. The site 1 serine to ala nine mutant was phosphorylated at site 2, but phosphorylation had no e ffect upon enzyme activity. Mutation of site 2 serine to either glutam ate or alanine also had no effect upon enzyme activity, but phosphoryl ation of these proteins at site 1 inhibited enzyme activity. E1 mutate d to change both phosphorylation site serines to glutamates was withou t enzyme activity. The binding affinity of E1 to the E2 core was not a ffected by mutation of the phosphorylation sites to glutamates, sugges ting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphor ylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphoryla tion.