Effect of reductive alkylation on catalytic properties of glycogen phosphorylase B. enzyme derivatives with changed nucleotide affinities.

Glycogen phosphorylase b modified by NaBH4 and aliphatic aldehydes of varying chain length: ranging from acetaldehyde to n-heptanaldehyde were purified by heat-treatment. Kinetic studies showed that the various purified enzyme derivatives were similar to native phosphorylase b in kinetic properties with respect to glucose-I-P. They, however, exhibited different affinities toward AMP. For acetaldehyde, propionaldehyde and butyraldehyde modified phosphorylases b, increase in chain length of the modifying aldehyde resulted in a decrease in AMP affinity of the modified enzyme. For the other aldehydes increase in chain length resulted in an increased AMP affinity of the modified enzyme. Consequently, the apparent values of butyraldehyde and heptanaldehyde modified phosphorylase b exhibited greatest deviation from that of native phosphorylase b. These two enzyme derivatives were studied in more detail. The activation of both enzyme derivatives by AMP could be greatly stimulated by spermine at suboptimal levels of AMP. Both derivatives could be activated by IMP, UMP or CMP in addition to AMP. The extents of activation of heptanaldehyde modified phosphorylase b by these nucleotides were higher than those found for native phosphorylase b. Thus, under certain conditions which included using suboptimal AMP or using IMP, UMP or GMP instead of AMP, heptanaldehyde modified phosphorylase b had much higher catalytic activity than native phosphorylase b. The interactions between burtyraldehyde or heptanaldehyde modified phosphorylases b with IMP were respectively weaker or stronger than that between native phosphorylase b and IMP. The interactions between butyraldehyde or heptanaldehyde modified phosphorylase b with glucose-6-P, an inhibitor of phosphorylase b partially competitive with respective to AMP, were also respectively weaker or stronger than that between the inhibitor and the native enzyme.