STEREOSPECIFIC REQUIREMENTS FOR CARBONYL REAGENTS IN RESOLUTION AND RECONSTITUTION OF PHOSPHORYLASE B

Removal of pyridoxal 5′-phosphate from phosphorylase by means of deforming agents and carbonyl reagents is a stereospecific process. Resolution occurs with lcysteine but not with D-cysteine, although the two isomers have identical association constants with pyridoxal 5′-phosphate. D-Cysteine does not compete with L-cysteine in the removal of pyridoxal 5′-phosphate from the enzyme. Stereospecificity also is observed in the reconstitution of apophosphorylase b with pyridoxal 5′-phosphate to yield the holoenzyme; however, this reaction is inhibited by the presence of a large excess of D-cysteine due to competition of the two en- antiomorphs for the free cofactor. Evidence is presented for the existence of a cysteine binding site in phosphorylase. Upon incubation of the enzyme with radioactively labeled cysteine and gel filtration on Sephadex G-25, binding of ca. 1 mole of cysteine per mole of enzyme protomer (mol wt 92,500) was observed. The bound cysteine can be removed by exhaustive dialysis or urea denaturation indicating that it is probably linked to the protein by a noncovalent bond or a bond that is easily hydrolyzed. Data indicate that during both resolution and reconstitution of phosphorylase the cofactor leaves and reenters the protein as a pyridoxal 5′-phosphate-L-cysteine complex. © 1969, American Chemical Society. All rights reserved.