Physicochemical changes in phosphorylase kinase associated with its activation.
Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca(2+)-dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as upward arrow pH), but Ca(2+) dependence is retained. Little is known about the structural changes associated with PhK's activation by upward arrow pH and Ca(2+), but activation by both mechanisms is mediated through regulatory subunits of the (alphabetagammadelta)(4) PhK complex. In this study, changes in the structure of PhK induced by upward arrow pH and Ca(2+) were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy, and zeta potential analyses. The joint effects of Ca(2+) and upward arrow pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at pH approximately 7.6. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 - Ca(2+)) versus that where it would be most active (pH 8.2 + Ca(2+)), the joint activation by upward arrow pH and Ca(2+) is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of upward arrow pH and Ca(2+) in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.
Liu
Weiya
W
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Priddy
Timothy S
TS
Carlson
Gerald M
GM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D004789:Enzyme Activation; D005260:Female; D006863:Hydrogen-Ion Concentration; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D013499:Surface Properties
D010764:Phosphorylase Kinase; D002118:Calcium
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