Dataset Information

Glucoamylase-like domains in the alpha- and beta-subunits of phosphorylase kinase.

ABSTRACT: Phosphorylase kinase is a four-subunit enzyme involved in the regulation of glycogen breakdown. The traditional textbook view is that only the gamma subunit has enzymatic activity, whereas the other three subunits have a regulatory role. Evidence from homology searches and sequence alignments, however, shows that the alpha- and beta-subunits possess amino-terminal glucoamylase-like domains and suggests that they might possess a previously overlooked amylase activity. If true, this would have important implications for the understanding, diagnosis, and management of glycogen storage diseases. There is thus a clear need to test this hypothesis through enzymatic assays and structural studies.

SUBMITTER: Pallen MJ

PROVIDER: S-EPMC2323967 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Glucoamylase-like domains in the alpha- and beta-subunits of phosphorylase kinase.

Pallen Mark J MJ

Protein science : a publication of the Protein Society 20030801 8

Phosphorylase kinase is a four-subunit enzyme involved in the regulation of glycogen breakdown. The traditional textbook view is that only the gamma subunit has enzymatic activity, whereas the other three subunits have a regulatory role. Evidence from homology searches and sequence alignments, however, shows that the alpha- and beta-subunits possess amino-terminal glucoamylase-like domains and suggests that they might possess a previously overlooked amylase activity. If true, this would have imp ...[more]

PMID: 12876330

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Project description:Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.

Dataset Information

Glucoamylase-like domains in the alpha- and beta-subunits of phosphorylase kinase.

Publications

Glucoamylase-like domains in the alpha- and beta-subunits of phosphorylase kinase.

Similar Datasets

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