Cyclin-dependent kinase 2 (CDK2) is an important member of the CDK family exerting its most important function in the regulation of the cell cycle. It catalyzes the transfer of the gamma phosphate group from an ATP (adenosine triphosphate) molecule to a Serine/Threonine residue of a peptide substrate. Due to the importance of this enzyme, and protein kinases in general, a detailed understanding of the reaction mechanism is desired. Thus, in this work the phosphoryl transfer reaction catalyzed by CDK2 was revisited and studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that the base-assisted mechanism is preferred over the substrate-assisted pathway when one Mg 2+ is present in the active site, in agreement with a previous theoretical study. The base-assisted mechanism resulted to be dissociative, with a potential energy barrier of 14.3 kcal/mol, very close to the experimental derived value. An interesting feature of the mechanism is the proton transfer from Lys129 to the phosphoryl group at the second transition state, event that could be helping in neutralizing the charge on the phosphoryl group upon the absence of a second Mg 2+ ion. Furthermore, important insights into the mechanisms in terms of bond order and charge analysis were provided. These descriptors helped to characterize the synchronicity of bond forming and breaking events, and to characterize charge transfer effects. Local interactions at the active site are key to modulate the charge distribution on the phosphoryl group and therefore alter its reactivity.

Phosphofructokinases catalyze the ATP-dependent phosphorylation of fructose-6-phosphate and they are highly regulated.

Lignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzyme with its substrates is essential in the rational design of engineered peroxidases for biotechnological application. Here, we combine computational and experimental approaches to analyze the interaction of Phanerochaete chrysosporium LiP (isoenzyme H8) with VA and its radical cation (VA •+ , resulting from substrate oxidation by the enzyme). Interaction energy calculations at semiempirical quantum mechanical level (SQM) between LiP and VA/VA •+ enabled to identify those residues at the acidic environment of catalytic Trp171 involved in the main interactions. Then, a battery of variants, with single and multiple mutations at these residues (Glu168, Asp165, Glu250, Asp264, and Phe267), was generated by directed mutagenesis, and their kinetics parameters were estimated on VA and two additional substrates. The experimental results show that Glu168 and Glu250 are crucial for the binding of VA, with Glu250 also contributing to the turnover of the enzyme. The experimental results were further rationalized through new calculations of interaction energies between VA/VA •+ and LiP with each of the single mutations. Finally, the delocalization of spin density was determined with quantum mechanics/molecular mechanics calculations (QM/MM), further supporting the contribution of Glu250 to VA oxidation at Trp171.

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