Regulation of Human Serine Racemase Activity and Dynamics by Halides, ATP and Malonate

Online resources 1

Regulation of human serine racemase activity and

dynamics by halides, ATP and malonate

Marialaura Marchetti, Stefano Bruno, Barbara Campanini,

Stefano Bettati, Alessio Peracchi and Andrea Mozzarelli

1 Materials and methods

Equation S1

Mixed static and dynamic quenching is described by equation S1:

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).

Equation S2

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).

where y is the fluorescence emission intensity, [P] is the fixed protein concentration and

[L] the variable ligand concentration. y0 is a an horizontal offset and a the amplitude.

Fig S1

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,

1 mM EDTA (TRIS + EDTA); 50 mM TEA (TEA), 50 mM TEA, 1 mM EDTA (TEA

+ EDTA), 50 mM sodium phosphate (Phosphate); 50 mM sodium phosphate, 1 mM

EDTA (Phosphate + EDTA); 50 mM TRIS plus 100 mM imidazole (TRIS + imidazole).

All solutions were at pH 8.0. The residual activity is expressed as a fraction of the

-1 -1 activity of the enzyme before dialysis, 1.8 μmolpyruvatemin mg .

Fig S2

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.

4 Fig S3

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

-1 normalized to a specific activity measured in the absence of salts of 2.3 μmolpyruvatemin mg-1.

5 Fig S4

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

-1 -1 specific activity measured in the absence of salts of 2.3 μmolpyruvatemin mg .

6 Fig S5

Fig S5 Fluorescence spectra of hSR were recorded in a solution containing 2.4 μM hSR,

50 mM TEA, 150 mM NaCl, 5 mM TCEP, 1 mM MgCl2, pH 8.0 at 20 °C.

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.

8 Fig S6

Fig S6 Effect of fluoride and chloride on the fluorescence emission spectra of hSR.

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

TCEP, 1 mM MgCl2, pH 8.0 at 20°C either in the absence (solid line) and presence

(dashed line) of 150 mM fluoride (panels A and B) or 150 mM chloride (panel C and

D). Slitexwas 5 nm and slitem was 5 nm

9 References

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.