The role of lysine 385 in yeast phosphoglycerate kinase

Site specific mutants K385E and K385T of yeast phosphoglycerate kinase were made and used in kinetic, CD, and NMR experiments. Lys385 is located on a β-tum at the end of an α-helix, near the metal-ATP binding site on the enzyme. A previous study showed that Lys385 was specifically modified by an ATP analog that contained a pyridoxal moiety in place of the y-phosphoryl group of ATP (Pineda et. al. (1993). Eur. J. Biochem. 212, 719-726). Kinetic studies showed K_M and k_cat values similar to those of the wild-type enzyme indicating that Lys385 is not catalytically required. However, substrate activation by ATP was altered with K385E when compared to the wild-type enzyme. Also, protection of the enzyme activity by ATP against heat inactivation was significantly reduced with K385E. K385T was the least stable enz\Tne against unfolding by guanidine hydrochloride while K385E showed similar behavior to the wild-type. ID ¹H NMR spectra showed that there was no observable chemical shift differences between the spectra of the mutants and wild-type enzyme. The enhancement of the paramagnetic effect of Mn²⁺ on water protons in the ternary enzyme-Mn²⁺-ATP complexes of both mutants was higher than for the wild-type, suggesting that ATP binding to the mutants is different compared to that of the wild-type. The above results suggest that Lys385 may be in or near the proposed secondary ATP substrate binding site and may be important to interdomain interactions that stabilize the protein.