Catalytic Mechanism of Phosphorylase Kinase Probed by Mutational Studies,

Abstract

The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1−298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K_m for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k_cat (1.3 × 10⁴ for Asp149Ala and 4.7 × 10³ for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 × 10³ and a significant decrease (possibly as great as 2.2 × 10³) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646−6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k_cat/K_m by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 Å resolution showed a phosphate dianion about 2.6 Å from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis.