Tyrosine analogues as alternative substrates for protein tyrosine kinase Csk: insights into substrate selectivity and catalytic mechanism

Protein Tyrosine Kinases are critical Enzymes in cell signal transduction but relatively little is known about the molecular recognition of the tyrosine substrate by these Enzymes. Details of tyrosine substrate specificity within the context of a short peptide were investigated for protein tyrosine kinase Csk. It was found that aryl ring functional group substitutions the size of methyl group or smaller were generally well tolerated by the protein tyrosine kinase Csk whereas larger groups caused a decline in substrate efficiency. Extension of the phenol from the peptide backbone by a single methylene was acceptable for phosphorylation whereas removal of a methylene nearly abolished reactivity. Only the L-tyrosine derivative was processed. A negative charge ortho to the phenol hydroxyl was incompatible with substrate reactivity, consistent with previous pH rate profiles which indicated the importance of the neutral phenol. Overall, these studies confirmed the interpretation of a previous linear free energy relationship analysis which suggested that the Enzyme followed a dissociative transition state mechanism.