Abstract
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 in K(m)((ATP)). K(m) for mevalonate increases by approximately 20-fold for S146A, approximately 40-fold for T243A, and 100-fold for S201A. V(max) changes for S145A, S201A, and T243A are < or =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 10(5)-fold effect, suggesting an active site location and catalytic role for Ser-146. The large k(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 Mg(2+)-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.
Highlights
Isoprenoid biosynthesis is accomplished by diverse pathways that use either acetyl-CoA [1] or glyceraldehyde 3-phosphate and pyruvate [2] as starting materials
The diversity of species in which mevalonate kinase functions is reflected in the high degree of heterology that is apparent upon comparison of deduced sequences for this enzyme
The results of this study contribute to the mapping of the active site and refine and expand the hypotheses that account for mevalonate kinase function and are likely to be relevant to other members of the GHMP kinase family
Summary
Isoprenoid biosynthesis is accomplished by diverse pathways that use either acetyl-CoA [1] or glyceraldehyde 3-phosphate and pyruvate [2] as starting materials. 5% of the amino acids that encode the human enzyme are invariant Included in this select group are the residues (Lys Glu-193, Asp-204) that our laboratory has implicated previously [6, 7] in various active site functions. We aligned and examined the available (Ͼ20) mevalonate kinase sequences and noted that only four residues that contain such side chains are invariant. Of these four residues, two map in one of two glycine-rich sequences proposed to be ATP-binding motifs. On the basis of these observations, we launched an investigation of the functional significance of mevalonate kinase’s invariant serine/threonine residues. The results of this study contribute to the mapping of the active site and refine and expand the hypotheses that account for mevalonate kinase function and are likely to be relevant to other members of the GHMP kinase family
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