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, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates 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 phosphorylation site 1 serine to glutamate inactivated E1 activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased Km for the alpha-ketoacid substrate but had no effect on maximum velocity. The site 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity, but phosphorylation of these proteins at site 1 inhibited enzyme activity. E1 mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E1 to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphorylation.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Y
Y
Zhao
J
J
Hawes
K M
KM
Popov
J
J
Jaskiewicz
Y
Y
Shimomura
D W
DW
Crabb
R A
RA
Harris
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000409:Alanine; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005822:Genetic Vectors; D005971:Glutamates; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity
D005971:Glutamates; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012694:Serine; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D011494:Protein Kinases; C035627:(3-methyl-2-oxobutanoate dehydrogenase (lipoamide)) kinase; D000409:Alanine