Abstract
Pyruvate kinase M1, a non-allosteric isozyme, was converted into an allosteric enzyme by replacement of an amino acid in the intersubunit contact. The substitution of Ala-398 with Arg resulted in the pronounced allosteric enzyme. The Hill coefficient and the substrate concentration giving one-half of Vmax for the mutant with respect to phosphoenolpyruvate were 2.7 and 0.41 mM, respectively, whereas those values for the wild type were 1.0 and 0.049 mM. This mutation, however, gave rise to only minor effects on the apparent values of Km for ADP and on Vmax. Furthermore, in contrast to the wild-type enzyme, the mutant was activated by fructose 1, 6-bisphosphate. The Hill coefficient of the mutant was no longer increased by the allosteric inhibitor, L-phenylalanine, indicating that the equilibrium for the unligated enzyme is largely shifted toward the T-state. These results suggest that Ala-398 is one of the most critical residues allowing the enzyme to prefer the R-state and that allosteric regulation of pyruvate kinase involves amino acid residues in the intersubunit contact.
Highlights
Pyruvate kinase M1, a non-allosteric isozyme, was converted into an allosteric enzyme by replacement of an amino acid in the intersubunit contact
We examined the effects of amino acid substitutions on the kinetic properties of the rat PK-M1 isozyme to identify the residues responsible for the enzyme to favor the R-state
Our results suggest that Ala-398 is a critical residue for the stabilization of the R-state in the PK-M1 isozyme
Summary
Materials—Restriction endonuclease and DNA-modifying enzymes were purchased from Takara. Preparation of Recombinant Viruses—The purified transfer plasmids containing the wild-type or mutant PK-M1 cDNA (1 g) were co-transfected into 5 ϫ 105 Sf21 cells with 10 ng of BaculoGold DNA (PharMingen), which was used as Autographa californica nuclear polyhedrosis viral genome. Enzyme Activity Assay—Standard assay for PK activity was performed at 37 °C using 2 mM both PEP and ADP in 50 mM Tris-HCl buffer, 0.1 M KCl, 5 mM MgSO4, and 0.5 mM FBP (pH 7.5) as described [1] This substrate mixture (1 ml) contained 17 units of lactate dehydrogenase and 0.17 mM NADH to monitor release of pyruvate as a change of absorbance at 340 nm.
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