Role of Arg228 in the Phosphorylation of Galactokinase: The Mechanism of GHMP Kinases by Quantum Mechanics/Molecular Mechanics Studies

Abstract

GHMP kinases are a group of structurally related small molecule kinases. They have been found in all kingdoms of life and are mostly responsible for catalyzing the ATP-dependent phosphorylation of intermediary metabolites. Although the GHMP kinases are of clinical, pharmaceutical, and biotechnological importance, the mechanism of GHMP kinases is controversial. A catalytic base mechanism was suggested for mevalonate kinase that has a structural feature of the γ-phosphate of ATP close to an aspartate residue; however, for one GHMP family member, homoserine kinase, where the residue acting as general base is absent, a direct phosphorylation mechanism was suggested. Furthermore, it was proposed by some authors that all the GHMP kinases function by a similar mechanism. This controversy in mechanism has limited our ability to exploit these enzymes as drug targets and in biotechnology. Here the phosphorylation reaction mechanism of the human galactokinase, a member of the GHMP kinase family, was investigated using molecular dynamics simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B3LYP-D/AMBER99). The reaction coordinates were localized by potential energy scan using an adiabatic mapping method. Our results indicate that a highly conserved Glu174 captures Arg105 in the proximity of the α-phosphate of ATP, forming a H-bond network; therefore, the mobility of ATP in the large oxyanion hole is restricted. Arg228 functions to stabilize the negative charge developed at the β,γbridging oxygen of the ATP during bond cleavage. The reaction occurs via a direct phosphorylation mechanism, and the Asp186 in the proximity of ATP does not directly participate in the reaction pathway. Because Arg228 is not conserved among GHMP kinases, reagents which form interactions with Arg228, and therefore can interrupt its function in phosphorylation, may be developed into potential selective inhibitors for galactokinase.

Phosphorylation Mechanism of Phosphomevalonate Kinase: Implications for Rational Engineering of Isoprenoid Biosynthetic Pathway Enzymes

“…10 Two conserved catalytic residues (Ser141 and Ser142) in galactokinase stabilize ATP in the oxyanion hole. Although the coordination environment of the Mg 2+ ion in the two GHMP kinases is different, we identified the corresponding functional residues in PMK by careful comparison of structures of galactokinase and PMK obtained from MD simulations.…”

“…45 GTMP kinases are different from classical kinases in that such a lysine is absent in the motif II p-loop, 10 except for PMK, which contains a lysine at the end of the p-loop. However, we have previously found that a positively charged residue (Arg228) in galactokinase can reorientate toward ATP to facilitate the phosphoryl transfer.…”

“…However, we have previously found that a positively charged residue (Arg228) in galactokinase can reorientate toward ATP to facilitate the phosphoryl transfer. 10 …”

Theoretical Studies of the Catalytic Mechanism of the Dihydroxyacetone Kinase

“…65,91,92,101 Here it will be presented theoretical studies in PKA, CDK2 and galactokinase (GALK) related with the finding of this mechanism. Some studies in the kinase PKA supported this mechanism in contrast to those mentioned above.…”

“…It should be remarked that Smith et al related this conclusion to the exclusion of the Asp from the QM region in the QM/MM simulations. Huang et al 101 located the substrate-assisted mechanism in GALK where the phosphoryl group is transferred from the ATP to the oxygen of one OH group of the substrate galactose. QM/MM simulations within a two-layer ONIOM method were carried out.…”