Desensitization to glucose 6-phosphate of phosphoenolpyruvate carboxylase from maize leaves by pyridoxal 5'-phosphate.
Incubation of the nonphosphorylated form of maize-leaf phospho enol pyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), PEPC, EC 4.1.1.31) with the reagent pyridoxal 5'-phosphate (PLP) resulted in time-dependent, reversible inactivation and desensitization to the activator glucose 6-phosphate (Glc6P) and other related phosphorylated compounds. Both processes are not connected, since (i) when the PLP-modification was carried out in the presence of saturating ligands of the active site, which prevents inactivation, the desensitization to Glc6P is still observed, and (ii) under some experimental conditions the desensitization reaction is 4-times faster than the inactivation. Desensitization to Glc6P is first order with respect to PLP and has a second-order forward rate constant of 4.7 +/- 0.3 s-1 M-1 and a first-order reverse rate constant of 0.0046 +/- 0.0002 s-1. Correlation studies between the remaining Glc6P sensitivity and mol of PLP residues incorporated per mol of enzyme subunit indicate that one lysyl group for enzyme monomer is involved in the sensitivity of the enzyme to Glc6P. The reactivity of this group is increased by polyethylene glycol and glycerol, while the reactivity of the lysyl group of the active site is not affected by these organic cosolutes. In the presence but not in the absence of the organic cosolutes, Glc6P by itself offers significant protection against desensitization, while increases the extent of inactivation. Free PEP or PEP-Mg have opposite effects, protecting the enzyme against inactivation and increasing the degree of desensitization. They also increases the protection against desensitization afforded by Glc6P. Finally, the PEPC inhibitor malate provides some protection against both inactivation and desensitization. Taken together, these results are consistent with PLP-modification of a highly reactive lysyl group at or near the allosteric Glc6P-site.
Tovar-Méndez
A
A
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México D.F., Mexico.
Mújica-Jiménez
C
C
Muñoz-Clares
R A
RA
D000495:Allosteric Site; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019298:Glucose-6-Phosphate / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D008293:Malates / Q000494:pharmacology; D015394:Molecular Structure; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D003313:Zea mays / Q000201:enzymology
D004791:Enzyme Inhibitors; D008024:Ligands; D008293:Malates; D019298:Glucose-6-Phosphate; D011732:Pyridoxal Phosphate; C030298:malic acid; D010730:Phosphoenolpyruvate Carboxylase