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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.

SUBMITTER: Sugden PH

PROVIDER: S-EPMC1161173 | biostudies-other | 1979 Aug

REPOSITORIES: biostudies-other

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Project description:1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...

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

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