Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione.
The dienophile 4-phenyl-1,2,4-triazoline-3,5-dione (PTD) was identified as a novel protein cross-linker, and utilized as a conformational probe of phosphorylase b kinase (PhK), a hexadecameric enzyme with the subunit composition (alphabetagammadelta)4. In its reaction with this enzyme, PTD produced five major cross-linked conjugates as resolved by denaturing gel electrophoresis: alphabeta, betagammagamma, alphagamma and a doublet of differently migrating homodimers, betabeta1 and betabeta2. Cross-linking in the presence of six different activators of the kinase targeted to its various subunits caused substantial changes in the amounts of three of the conjugates. The formation of alphagamma was increased by all of the activators but the largest enhancement was caused by exogenous Ca2+/calmodulin. All except one of the activators decreased the amount of betagammagamma formed, with Mg2+ having the greatest effect, and all except two increased the amount of betabeta1, with Mg2+ again having the largest influence. From the overall similarity of the changes in cross-linking by PTD induced by the various activators, we conclude that, even though they are targeted to different sites and subunits, they induce activated conformations of PhK that have certain structural features in common. Regarding the mechanism of cross-linking by PTD, its reaction with a model nucleophile suggests that its initial reaction with a side chain nucleophile of PhK involves a 1,4-conjugate addition to form a urazole adduct, with the secondary cross-linking reaction occurring through an as yet unknown pathway.
Ayers
N A
NA
Department of Biochemistry, College of Medicine, The University of Tennessee, Memphis, Memphis, TN 38163, USA.
Nadeau
O W
OW
Read
M W
MW
Ray
P
P
Carlson
G M
GM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013379:Substrate Specificity; D014230:Triazoles / Q000737:chemistry* / Q000378:metabolism
D002147:Calmodulin; D003432:Cross-Linking Reagents; D014230:Triazoles; C053540:4-phenyl-1,2,4-triazoline-3,5-dione; D010764:Phosphorylase Kinase; D002118:Calcium