The overall aim was to elucidate the substrate specificity and regulatory properties of the catalytic subunit of phosphorylase kinase (PhK) to better understand how this enzyme works. I have expressed the PhK [gamma] subunit (full-length and seven truncated forms) in E. coli. One of the truncated forms of [gamma], [gamma][subscript]1-300 has a 2-fold higher specific activity than the full-length [gamma], suggesting that an autoinhibitory domain(s) is located at the C-terminus of [gamma], [gamma][subscript]301-386. The truncated [gamma][subscript]1-300 purified to homogeneity has several properties similar to full-length [gamma], including its substrate specificity and metal ion response. Therefore, [gamma][subscript]1-300 was used as a model enzyme to probe structure-function relationships of PhK [gamma] subunit. Charge to alanine and charge reversal scanning mutations were used to locate substrate and metal ion binding sites of [gamma]. The secondary structures of mutant proteins were evaluated by FT-IR/PAS (Fourier transform infrared/photoacoustic spectroscopy). Those mutant proteins with similar secondary structures compared to wild-type [gamma][subscript]1-300 were further characterized. Two mutant proteins, E111K and E154R, were shown to be involved in substrate, pseudosubstrate, and metal ion binding. Using these two mutants, we demonstrated that E[superscript]111 binds to the P-3 site (K[superscript]11) and E[superscript]154 interacts with the P-2 site (Q[superscript]12) of phosphorylase b. Based on results with these two mutants and others, it is suggested that the second metal ion binding site of [gamma] is between the D[superscript]168FG loop and E[superscript]111--KPE[superscript]154N loop similar to the second metal ion binding site in cAMP-dependent protein kinase. Two synthetic peptides, PhK 13 ([gamma][subscript]302-326) and PhK 5 ([gamma][subscript]342-366), corresponding to two calmodulin binding regions of [gamma] were used as inhibitors. Based on studies of inhibition mechanisms of [gamma][subscript]1-300 and both mutant proteins, we suggest the inhibition mechanism of PhK 13 is through a pseudosubstrate mechanism and PhK 5 is via an inhibitory mechanism. The active site region of [gamma] is proposed to have similarities to calmodulin-binding site based on these data and other speculations. Because PhK and [gamma][subscript]1-300 can phosphorylate seryl and tyrosyl residues dependent on the metal ions, new substrates and pathways might exist for this enzyme beyond the known glycogenolysis cascade. Two proteins, which were phosphorylated on tyrosine in muscle cell extracts either directly or indirectly by PhK, raise this possibility.