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A substrate selective p38alpha inhibitor of mitogen-activated protein kinase (MK2a) phosphorylation is discovered. The CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.

Chicken liver pyruvate carboxylase is a tetrameric manganoprotein catalyzing a nonclassical ping-pong mechanism whereby biotin carboxylation at subsite 1 is coupled to biotin-dependent carboxylation of pyruvate at subsite 2. Simulation of oxaloacetate decarboxylase activity indicates that cofactors and substrates induce carboxybiotin movement from the second subsite to the first.

Conformational changes in the catalytic cycle of DNA polymerase beta were determined by stopped-flow fluorescence assay, utilizing a synthetic DNA primer/template with 2-aminopurine (2-AP) at the template position opposite the incoming dNTP. Two phases of fluorescence change were found in the stopped-flow fluorescence assay of the incorporation of the correct nucleotide dTTP. The rates of the two phases and their dependence on (dTTP) and (Mg2+) indicate that the fast conformational change is caused by the binding of MgdNTP and the slow conformational change is caused by the binding of the catalytic Mg2+ ion.

The kinetic parameters for all possible correct and incorrect pairing between the A, T, G and C bases were obtained for wild-type (WT) rat DNA polymerase beta (pol beta) and the R283A mutant under pre-steady-state kinetic assay conditions. Results indicated that for both WT and R283A, the fidelity is determined primarily by kpol (decreases for the incorporation of incorrect nucleotides). In general, the fidelity for the Y*X mismatch is different from that of the X*Y mismatch. It was also determined that the fidelity of R283A is reduced in all 12 mispairs compared to that of WT and the decreases in the fidelity of the R283A mutant are mainly due to alterations in the values of kpol.

The kinetics of DNA polymerase (pol) beta were investigated by rapid-chemical-quench and DNA trapping experiments. The results revealed that catalysis by pol beta is progressive and begins rapidly under pres-steady state conditions. However, pol beta had low fidelity and processivity as compared to other polymerases. Meanwhile, site-directed mutagenesis revealed that arginine-283 is not important to the stability and function of DNA pol beta.

Downstream signaling complexes and molecular mechanisms are analyzed, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. The 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex.

The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.