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The Activity of Phosphorylase Kinase Revisited
Dillon, Jackie Ann
Dillon, Jackie Ann
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Abstract
Phosphorylase Kinase (PhK) is an essential regulatory enzyme in the glycogenolysis cascade. PhK is a large, 16-subunit enzyme complex with the subunit stoichiometry (alpha-beta-gamma-delta)4, and is subject to extensive regulation by small molecules and reversible phosphorylation. Because of its large mass of 1.3 MDa, only limited experimental tools are available to study the structure of PhK and elucidate the structural changes that occur within the complex concomitant with activation. Therefore, our understanding of the roles of PhK’s subunits in regulating its kinase activity is woefully incomplete. The goal of this work was to revisit several aspects of PhK’s activity and further explore the roles of the enzyme’s individual subunits. Chemical crosslinking was the principal technique used in these studies because it is well-suited to study large, multi-subunit complexes. Using chemical crosslinking, plus additional methods, three major discoveries are presented regarding PhK’s activation and substrate recognition. First, zero-length oxidative crosslinking was used to selectively study conformational changes in the regulatory beta subunits, which led to the formulation of a model for activation of PhK. The model proposes that modification of the N-terminus of beta is key to activation of the catalytic gamma subunit and to conformational changes in the beta subunits, which likely cause global structural changes in the complex. Secondly, a two-step crosslinking approach revealed novel interactions between the regulatory alpha and beta subunits and PhK’s substrate, glycogen phosphorylase, establishing the first direct evidence of substrate binding sites on the regulatory subunits of the enzyme. Lastly, a temperature-dependent conformational change in the beta and gamma subunits was discovered to occur between the standard assay temperature of 30 °C and the physiological temperature of 40 °C. This temperature-dependent conformational change coincides with a surprising activation of PhK at physiological temperature. Steady progress continues to be made in studying the PhK complex. While the picture is still far from complete, this work contributes important details regarding the alpha, beta, and gamma subunits and their expanded roles in PhK’s activation and substrate interaction. Further exploration of the structure and activity of PhK is necessary to fully understand its critical function in glycogenolysis.
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Date
2018-05-31
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University of Kansas
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Keywords
Biochemistry, Chemical Crosslinking, Glycogenolysis, Glycogen Phosphorylase, Kinase, Kinetics, Phosphorylase Kinase