Component requirements for NADH inhibition and spermine stimulation of pyruvate dehydrogenaseb phosphatase activity.
The subunit and subdomain requirements for NADH inhibition as well as Ca+ and spermine activation of the pyruvate dehydrogenaseb phosphatase were analyzed. The transacetylase-protein X subcomplex (E2-X) was required for all three effects. The oligomeric inner domain of the transacetylase did not support any of these regulatory effects. The presence of at least a portion of the outer (lipoyl-bearing) domains of the transacetylase but not the lipoyl-bearing portion of protein X was essential for expression of these regulatory effects on phosphatase activity. The inner domain of protein X may contribute to some effects. The E2-X subcomplex, alone, had no effect on phosphatase activity in the absence of Ca2+, but the subcomplex did support both NADH inhibition and spermine activation in the absence of Ca2+. Studies with peptide substrates established that spermine is directly bound by a phosphatase subunit. With the resolved pyruvate dehydrogenase component (E1b) used as the substrate, the E2-X subcomplex transformed the effect of spermine from inhibiting to stimulating the rate of dephosphorylation by the phosphatase. The above observations suggest that binding of E1b to the E2-X subcomplex alters its presentation to the phosphatase. We also present several observations that are consistent with NADH inhibition of the phosphatase being mediated through a dihydrolipoyl dehydrogenase-dependent reduction of lipoyl moieties in the E2-X subcomplex. Overall, our data establish that the outer, lipoyl-bearing domains of the oligomeric transacetylase core have an essential role in the function and regulation of the pyruvate dehydrogenase phosphatase.
Rahmatullah
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Department of Biochemistry, Kansas State University, Manhattan 66506.
Roche
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D000478:Alkylation; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000378:metabolism*; D013096:Spermine / Q000494:pharmacology*; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000494:pharmacology
D009243:NAD; D013096:Spermine; C007409:dihydrolipoamide; D008063:Thioctic Acid; D010749:Phosphoprotein Phosphatases; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase; D005033:Ethylmaleimide; D002118:Calcium