<p>Online resources 1</p><p>Regulation of human serine racemase activity and</p><p> dynamics by halides, ATP and malonate</p><p>Marialaura Marchetti, Stefano Bruno, Barbara Campanini, </p><p>Stefano Bettati, Alessio Peracchi and Andrea Mozzarelli</p><p>1 Materials and methods</p><p>Equation S1</p><p>Mixed static and dynamic quenching is described by equation S1: </p><p> where F0, F, KSV and Q are as described in equation 1 (Materials and Methods) and V is a static quenching constant that represents an active volume element surrounding the excited fluorophore (1).</p><p>Equation S2</p><p>The dependence of the fluorescence emission intensity on ATP concentration in the presence of malonate was fitted to the equation for tight binding S2 (2).</p><p> where y is the fluorescence emission intensity, [P] is the fixed protein concentration and</p><p>[L] the variable ligand concentration. y0 is a an horizontal offset and a the amplitude. </p><p>Fig S1</p><p>2 Fig S1Residual β-elimination activity of hSR stored in the presence of 1 mM EDTA after cycles of dialysis versus solutions containing 50 mM TRIS (TRIS); 50 mM TRIS,</p><p>1 mM EDTA (TRIS + EDTA); 50 mM TEA (TEA), 50 mM TEA, 1 mM EDTA (TEA</p><p>+ EDTA), 50 mM sodium phosphate (Phosphate); 50 mM sodium phosphate, 1 mM</p><p>EDTA (Phosphate + EDTA); 50 mM TRIS plus 100 mM imidazole (TRIS + imidazole).</p><p>All solutions were at pH 8.0. The residual activity is expressed as a fraction of the</p><p>-1 -1 activity of the enzyme before dialysis, 1.8 μmolpyruvatemin mg .</p><p>Fig S2</p><p>3 Fig S2 Activity of hSR in the presence of 5 mM sodium pyrophosphate (PP) and in the absence and presence of increasing concentrations of ATP (A) or Mg2+ (B). The reference is the assay carried out in the absence of sodium pyrophosphate.</p><p>4 Fig S3</p><p>Fig S3 β-elimination activity of hSR in assay solution in AS (minus NaCl) with L- serine at 100 mM concentration. Assays were carried out in the absence (reference) and in the presence of 150 mM LiCl, KCl, NMe3·HCl and NH4Cl. All activities were</p><p>-1 normalized to a specific activity measured in the absence of salts of 2.3 μmolpyruvatemin mg-1.</p><p>5 Fig S4</p><p>Fig S4 Relative activity of hSR in the absence (Reference) and in the presence of 93 mM NaF, NaCl, NaI either in the presence (A) or absence (B) of 2 mM ATP. The assays were carried out with 100 mM L-serine. All activities were normalized to a</p><p>-1 -1 specific activity measured in the absence of salts of 2.3 μmolpyruvatemin mg .</p><p>6 Fig S5</p><p>Fig S5 Fluorescence spectra of hSR were recorded in a solution containing 2.4 μM hSR,</p><p>50 mM TEA, 150 mM NaCl, 5 mM TCEP, 1 mM MgCl2, pH 8.0 at 20 °C.</p><p>7 A. Fluorescence emission spectrum upon excitation at 298 nm. B. Fluorescence emission spectrum upon excitation at 412 nm. C. Fluorescence excitation spectrum for emission at 500 nm. Slitex was 5 nm and slitem was 5 nm.</p><p>8 Fig S6</p><p>Fig S6 Effect of fluoride and chloride on the fluorescence emission spectra of hSR.</p><p>Fluorescence emission spectra, upon excitation at 298 nm (panels A and C) and at 412 nm (panels B and D) of a solution containing 2.4 μM hSR in 50 mM TEA, 5 mM</p><p>TCEP, 1 mM MgCl2, pH 8.0 at 20°C either in the absence (solid line) and presence</p><p>(dashed line) of 150 mM fluoride (panels A and B) or 150 mM chloride (panel C and</p><p>D). Slitexwas 5 nm and slitem was 5 nm</p><p>9 References</p><p>1. Eftink, M. R., and Ghiron, C. A. (1981) Fluorescence quenching studies with proteins, Analytical biochemistry 114, 199-227. 2. Copeland, R. A. (2000) Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis, Wiley-VCH, Inc.</p><p>10</p>