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Catalytic Mechanism of Phosphorylase Kinase Probed by Mutational Studies

Lookup NU author(s): Professor Martin Noble

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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 x 10(4) for Asp149Ala and 4.7 x 10(3) 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 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) 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 Angstrom resolution showed a phosphate dianion about 2.6 Angstrom 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.


Publication metadata

Author(s): Skamnaki VT, Owen DJ, Noble MEM, Lowe ED, Lowe G, Oikonomakos NG, Johnson LN

Publication type: Article

Publication status: Published

Journal: Biochemistry

Year: 1999

Volume: 38

Issue: 4

Pages: 14718-14730

Print publication date: 14/10/1999

ISSN (print): 0006-2960

ISSN (electronic): 1520-4995

Publisher: American Chemical Society

URL: http://dx.doi.org/10.1021/bi991454f

DOI: 10.1021/bi991454f


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