Investigation of Invariant Serine/Threonine Residues in Mevalonate Kinase

Mevalonate kinase serine/threonine residues have been implicated in substrate binding and inherited metabolic disease. Alignment of >20 mevalonate kinase sequences indicates that Ser-145, Ser-146, Ser-201, and Thr-243 are the only invariant residues with alcohol side chains. These residues have been individually mutated to alanine. Structural integrity of the mutants has been demonstrated by binding studies using fluorescent and spin-labeled ATP analogs. Kinetic characterization of the mutants indicates only modest changes inK_m(ATP). K_m for mevalonate increases by ≈20-fold for S146A, ≈40-fold for T243A, and 100-fold for S201A. V_max changes for S145A, S201A, and T243A are ≤3-fold. Thus, the 65-fold activity decrease associated with the inherited human T243I mutation seems attributable to the nonconservative substitution rather than any critical catalytic function. V_max for S146A is diminished by 4000-fold. In terms of V/K_MVA, this substitution produces a 10⁵-fold effect, suggesting an active site location and catalytic role for Ser-146. The largek_cat effect suggests that Ser-146 productively orients ATP during catalysis. K_D(Mg-ATP)increases by almost 40-fold for S146A, indicating a specific role for Ser-146 in liganding Mg²⁺-ATP. Instead of mapping within a proposed C-terminal ATP binding motif, Ser-146 is situated in a centrally located motif, which characterizes the galactokinase/homoserine kinase/ mevalonate kinase/phosphomevalonate kinase protein family. These observations represent the first functional demonstration that this region is part of the active site in these related phosphotransferases.