Amino acid sequences around the sites of phosphorylation in the pig heart pyruvate dehydrogenase complex

Abstract

1. When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [gamma-32P]ATP by its intrinsic kinase, three phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. 2. When phosphorylated to inactivation by repetitive additions of limiting quantities of [gamma-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in activation of pyruvate dehydrogenase. 3. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [gamma-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. 4. Incubation of pyruvate dehydrogenase with excess [gamma-32P]ATP with termination of phosphorylation at about 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 sequence 1 contained about 1% total P. 5. These results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.

Role of individual phosphorylation sites in inactivation of pyruvate dehydrogenase complex in rat heart mitochondria

“…Phosphopeptide TA12 (rat or pig heart) gave with formic acid near-equal amounts of two phosphopeptides TF1 and TF2 (ratio TF1/TF2, 0.94-0.99). Pig heart complex TF1 contains 32p in (Sugden et al, 1979). TF1 is residues 1-8 of TAI or TA12 and TF2 is residues 9-14 of TA2 or TA12 formed by formic acid cleavage of Asp8-Pro9.…”

“…Sale & Randle (1981b) have recently overcome this problem for pig heart complex by combining established methods of tryptic digestion (Davis et al, 1977) with formic acid cleavage. Trypsin yields a tetradecapeptide (TA) containing sites 1 (Ser-5) and 2 [sequence 2 of Sugden et al (1979) for pig heart complex] and a nonapeptide (TB) containing site 3 [sequence 1 of Sugden et al (1979)] (see also the legend to Table 1). Three TA [32P]phosphopeptides may be produced, TAI (32P at Ser-5), TA2 (32P at Ser-12) and TA 12; TA1 and TA2 coelectrophorese.…”

The pyruvate dehydrogenase complex of Saccharomyces cerevisiae is regulated by phosphorylation

“…The two prominent polypeptides observed in lanes 1 and 2 are presumed to represent the Elc~ subunits of the pyruvate and branched-chain 2-oxo acid dehydrogenase multienzyme complexes (PDC and BCOADC). These radiolabelled components with Mr values 41,000 and 46,000, respectively, have been previously identified as the only two reversibly phosphorylated proteins known to be present in mammalian mitochondria [3][4][5]14]. Both PDC and BCOADC contain distinct intrinsic kinases which co-purify with the complexes, inactivating them in an ATP-dependent reaction.…”

“…Phosphorylation by PDC kinase inactivates the complex, while dephosphorylation by a specific PDC phosphatase results in its reactivation. The phosphorylation sites in mammalian PDC are located on three particular serine residues in the ~ subunit of the pyruvate dehydrogenase (El) component of the complex [4][5][6].…”

Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex

“…is phosphorylated and inactivated by an intrinsic kinase (Linn et al, 1969a,b.). In complexes from bovine kidney or heart, or pig heart, sequence analyses of tryptic phosphopeptides have shown that inactivation is correlated with phosphorylation of one specific serine residue of the a-chain of the decarboxylase (EC 1.2.4.1) (Yeaman et al, 1978; the preceding paper, Sugden et al, 1979). The decarboxylase is a tetramer (a2,f2) (Barrera et al, 1972), and, in the pig heart complex, inactivation may be represented by the equation (a2fi2).+nMgATP -* (aP a/2)n+nMgADP (1) There is evidence for a similar stoicheiometry of inactivation by phosphorylation in bovine kidney and heart complexes (Yeaman et al, 1978).…”