Convergent and divergent evolution of regulatory sites in eukaryotic phosphorylases

Abstract

The activity of many proteins in eukaryotic cells is regulated by reversible covalent phosphorylation¹. This regulatory modification is often linked to other allosteric controls within the same protein², and such overlapping regulatory mechanisms are best characterized for glycogen phosphorylase (EC 2.4.1.1). Phosphorylases from different organisms or cell types exhibit markedly contrasting regulatory features³; this makes the enzyme attractive for studying the evolution of interacting molecular regulatory mechanisms^4,5. Extensive biochemical and crystallographic studies of rabbit muscle phosphorylase have led to a characterization of five regulatory regions (phosphorylation, glycogen storage, AMP, glucose and purine sites)^6–8. Here we report the complete primary structure of the yeast Saccharomyces cerevisiae glycogen phosphorylase, deduced from the sequence of the cloned gene. Regions that are highly conserved between muscle and yeast enzymes include the active site, the glycogen storage site and possibly the glucose and purine inhibition sites. Partial conservation of the residues involved in AMP-binding suggests a binding site for the yeast enzyme inhibitor, glucose 6-phosphate^9,10. Other parts of the AMP site and the intersubunit contacts involved in AMP allostery are disrupted in the yeast enzyme by extreme sequence divergence. The poor alignment of amino termini and lack of homology at phosphorylation sites indicate that regulation by reversible phosphorylation evolved independently in yeast and vertebrate phosphorylases.