Interaction of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase with pyridoxal 5'-diphospho-5'-adenosine. Affinity labeling of Lys-21 and Lys-343.
Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides competitively with respect to glucose 6-phosphate and noncompetitively with respect to NAD+ or NADP+, with Ki = 40 microM in the NADP-linked and 34 microM in the NAD-linked reaction. Incubation of glucose-6-phosphate dehydrogenase with [3H]PLP-AMP followed by borohydride reduction shows that incorporation of 0.85 mol of PLP-AMP per mol of enzyme subunit is required for complete inactivation. Both glucose 6-phosphate and NAD+ protect against this covalent modification. The proteolysis of the modified enzyme and isolation and sequencing of the labeled peptides revealed that Lys-21 and Lys-343 are the sites of PLP-AMP interaction and that glucose 6-phosphate and NAD+ protect both lysyl residues against modification. Pyridoxal 5'-phosphate (PLP) also modifies Lys-21 and probably Lys-343. Lys-21 is part of a highly conserved region that is present in all glucose-6-phosphate dehydrogenases that have been sequenced. Lys-343 corresponds to an arginyl residue in other glucose-6-phosphate dehydrogenases and is in a region that is less homologous with those enzymes. PLP-AMP and PLP are believed to interact with L. mesenteroides glucose-6-phosphate dehydrogenase at the glucose 6-phosphate binding site. Simultaneous binding of NAD+ induces conformational changes (Kurlandsky, S. B., Hilburger, A. C., and Levy, H. R. (1988) Arch. Biochem. Biophys. 264, 93-102) that are postulated to interfere with Schiff's-base formation with PLP or PLP-AMP. One or both of the lysyl residues covalently modified by PLP or PLP-AMP may be located in regions of the enzyme undergoing the NAD(+)-induced conformational changes.
LaDine
J R
JR
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210.
Carlow
D
D
Lee
W T
WT
Cross
R L
RL
Flynn
T G
TG
Levy
H R
HR
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D002851:Chromatography, High Pressure Liquid; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives*; D013379:Substrate Specificity
D000345:Affinity Labels; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D005954:Glucosephosphate Dehydrogenase; C048236:pyridoxal 5'-diphospho-5'-adenosine