Functional role of dimerization of human peptidylarginine deiminase 4 (PAD4).

Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca²âº-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (K(d)) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The K(d) values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower k(cat) values than that of WT (13.4 s⁻¹). The K(d) values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the k(cat) values of the monomeric mutants ranged from 3.3 to 7.3 s⁻¹. The k(cat) values of these interface mutants decreased as the K(d) values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation.

Functional role of fumarate site Glu59 involved in allosteric regulation and subunit-subunit interaction of human mitochondrial NAD(P)+-dependent malic enzyme.

Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME). In the present study, Glu59 was substituted by Asp, Gln or Leu. Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme. The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, K(A,Fum) and alphaK(A,Fum). The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activation. The K(A,Fum) and alphaK(A,Fum) of E59D are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding. Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer-tetramer equilibrium. In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59. The E59L enzyme also shows a dimer-tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers. Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate-binding site. Glu59 can precisely maintain the geometric specificity for the substrate cooperativity. According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation. Glu59 discriminates human m-NAD-ME from mitochondrial NADP+-dependent malic enzyme and cytosolic NADP+-dependent malic enzyme in fumarate activation and malate cooperativity.