David B. Glass

Substrate diversity of the cAMP-dependent protein kinase: regulation based upon multiple binding interactions

The proposition is forwarded that the cAMP-dependent protein kinase is one of quite a small class of enzymes wherein differential modes of binding of its multiple substrates make an important contribution to the end physiological response. It is postulated that a variety of different substrate affinities may have evolved in order to regulate the order of substrate phosphorylation. The recent elucidation of the proteins three-dimensional structure provides the opening to test this as a new concept of cellular regulation.

A [3H] lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.

In vitro Phosphorylation of a Synthetic Collagen Peptide by Cyclic AMP-Dependent Protein Kinase

A synthetic tridecapeptide that corresponds closely to amino acid residues 98 to 110 in chick collagen alpha 1(I) contains several determinants of specificity required for recognition and phosphorylation by the cyclic AMP-dependent protein kinase. The peptide Gly-Leu-Hyp-Gly-Nle-Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was predicted to be a substrate for the cyclic AMP-dependent protein kinase because it contained multiple basic amino acids NH2-terminal to a potentially phosphorylatable seryl residue. When tested as a substrate for the enzyme, the peptide was stoichiometrically phosphorylated. Phosphoserine was identified as the only phosphoamino acid in a partial hydrolysate of the phosphorylated peptide. The peptide and several of its analogs were phosphorylated by the cyclic AMP-dependent protein kinase with Km values from 1 to 6 mM and Vmax values from 1 to 3 mumol of phosphate/min/mg of enzyme. Although the Km of the kinase for the collagen peptide was high, these results confirmed the prediction made from knowledge of the substrate specificity of cyclic AMP-dependent protein kinase. The potential for such a phosphorylation reaction to occur in vivo is discussed.