This journal is The Royal Society of Chemistry 25 Supplementary data Inactivation of Human Angiotensin Converting Enzyme by Copper Peptide Complexes Containing ATCUN Motifs. Nikhil H. Gokhale and J. A. Cowan* Evans Laboratory of Chemistry, Ohio State University, 1 West 18 th Columbus, Ohio 4321 Avenue, SUPPORTING MATERIAL 1. Materials and Methods 1.1 Determination of Kinetic Constants. Determination of K m and V max values was carried out using the same fluorescence assay as described earlier. Reactions were carried out for 3 min at 37 C. Initial rates for the hydrolysis of substrate (V o ) were determined by following the change in fluorescence (relative fluorescence units/min, /min), plotted as a function of substrate concentration ([S]) and fit to the Michaelis-Menten equation V o = V max [S]/ ([S]+K m ), using Origin software (Microcal) to determine K m. Substrate concentration was varied from to 2 µm (Figure SM1 to SM4). 1.2 Determination of IC 5 values for rhace inhibitors using, Mca-Arg-Pro-Pro- Gly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH substrate. Reaction mixtures (1 µl) contained 5 mm HEPES buffer, 3 mm NaCl and 1 µm ZnCl 2 (ph 7.4), 1 µm substrate and 1nM (13.1 ng) rhace. Concentrations of GGH, KGHK, YIHPF, Cu 2+, Cu(GGH) -, Cu(KGHK) +, Cu(YIHPF) -, were varied from to 1 µm. Prior to the start of the enzymatic reaction (by addition of substrate) the inhibitors were pre-incubated with the enzyme for 45 min. Reactions were run in the wells of polystyrene 96-well microplates (Porvair) as described previously. Fluorescence was measured for 7 min. and the background rate determined for samples containing no rhace was subtracted from all reactions to calculate the initial rates in /min. Initial rate data were plotted as percentage activity, relative to uninhibited control reactions, as a function of inhibitor concentration (Figure SM5 to SM9). 1.3 Determination of the Type of Inhibition. Reaction mixtures (1 µl) contained 5 mm HEPES buffer, 3 mm NaCl, 1 µm ZnCl 2 (ph 7.4) and 1nM (13.1 ng) rhace. Two sets of reactions were performed at distinct substrate concentration (5 and 1 µm) with varying concentrations of inhibitor ( to 2 µm). Prior to the start of the enzymatic reaction (by addition of substrate) the inhibitors were pre-incubated with the enzyme for 45 min. Reactions were run in the wells of polystyrene 96-well microplates (Porvair). Fluorescence change was measured for 3 min as described previously. Initial rate data were plotted versus inhibitor concentration and a Dixon plot (inverse of initial velocity versus inhibitor concentration) was generated using Origin (Microcal). 1.4 Determination of Inhibition Constants. For the competitive inhibitors the enzyme-inhibitor dissociation constant (K I ) was determined by graphical methods by use
This journal is The Royal Society of Chemistry 25 of a Dixon plot, as well as by the equation below, where IC 5, [S] and K m have units of molar concentration. K I = IC 5 / (1+[S]/K m ) 1.5 Determination of the Inhibition Mechanism under Oxidative Reaction Conditions. Enzymatic reactions performed in the presence of dioxygen and ascorbate are defined as oxidative conditions, while all other reactions described herein that contained no ascorbate are defined as hydrolytic. Reaction mixtures (1 µl) contained 5 mm HEPES buffer, 3 mm NaCl, 1 µm ZnCl 2 (ph 7.4), 1 µm ascorbate (Lascorbic acid, prepared in deionized water) and 1 nm (13.1 ng) rhace. The concentration of inhibitor was set at K I. Prior to the start of the enzymatic reaction (either by addition of substrate or the enzyme) the inhibitors were pre-incubated with the enzyme for 45 min. Two sets of reactions were run in the wells of polystyrene 96-well microplates (Porvair) and fluorescence change (relative fluorescence units/min, /min) was measured for 3 min as described previously. Set I: Monitoring the progress curve for the enzymatic reaction under oxidative conditions: Following prior pre-incubation of the enzyme with the inhibitor, the enzymatic reaction was initiated by simultaneous addition of the substrate and ascorbate, and the progress of the reaction monitored by formation of product as reflected by the observed change of fluorescence. The initial velocity (V o ), obtained from the progress curve in the presence and absence of the inhibitor, was plotted versus time as described. Respective control reactions had either ascorbate present or absent (Figure SM1 and Figure SM11). Set II: Enzyme deactivation under oxidative conditions in the presence and absence of inhibitor: To obtain a true rate of enzyme deactivation by the inhibitor under oxidative conditions the enzyme was pre-incubated with the inhibitor for 45 min. Ascorbate was added, followed by a preincubation time, and aliquots of the reaction mixture were taken at various time intervals and the residual enzyme activity estimated using the 96-well plate assay described previously. Initial velocity (V o ) obtained in the presence and absence of inhibitor was plotted as a function of time and fitted to the first-order rate equation to obtain k obs.
This journal is The Royal Society of Chemistry 25 Figures for Supplementary Material. (background corrected) 6 5 4 3 2 1 5 µm Substrate 1 µm Substrate 2µM Substrate 3µM Substrate 4µM Substrate 5 µm Substrate Figure SM1 5 1 15 2 25 3 Time (MIn) 2 Initial Velocity (/Min) 15 1 5 1 2 3 4 5 Flurogenic Peptide substrate (µm) Figure SM2
This journal is The Royal Society of Chemistry 25 8 7 6 5 4 3 2 1 µm substrate 2µM substrate 4µM substrate 5µM substrate 8µM substrate 12µM substrate 14µM substrate 16µM substrate 18µM substrate 2µM substrate 1 2 3 4 5 Figure SM3.2.16 1/v ( -1.min).12.8-1/Km = -.15.4 K m = 6.6 µm. -.2 -.1..1.2.3.4.5 1/[s] (µm -1 ) Figure SM4
This journal is The Royal Society of Chemistry 25 Dose Dependence GGH Dose Dependence KGHK 2 18 16 14 12 1 8 6 µm 2 µm 4 µm 6 µm 8 µm 1 µm 2 µm 4 µm 8 µm 12 µm GGH 4 2 1 2 3 4 5 6 7 8 Time (min) 2 15 1 5 µm 4 µm 6 µm 8 µm 1 µm 2 µm 12 µm KGHK 1 2 3 4 5 6 7 8 Time (min) Dose Dependence YIHPF 25 2 15 1 5 µm 4 µm 6 µm 8 µm 1 µm 2 µm 8 µm 1 µm 12 µm YIHPF (initial velocity) 3. 2.5 2. 1.5 1..5 GGH KGHK YIHPF 1 2 3 4 5 6 7 8 Time (min). 1 1 1 Concentration (µm) Figure SM5
This journal is The Royal Society of Chemistry 25 For Cu 2+ Dose Dependance (Cu +2 ) 4 3 2 1 µm Cu +2 2 µm Cu +2 4 µm Cu +2 6 µm Cu +2 8 µm Cu +2 1 µm Cu +2 2 µm Cu +2 4 µm Cu +2 6 µm Cu +2 8 µm Cu +2 1 µm Cu +2 % Enzyme Activity 1 8 6 4 2 1 2 3 4 5 6 7 1 1 1 [I] (Cu +2 ) (µm) Figure SM6 For Cu(GGH) - 5 4 3 2 1 Dose Dependance Cu(GGH) + µm (GGH-Cu) -1 2 µm (GGH-Cu) -1 4 µm (GGH-Cu) -1 6 µm (GGH-Cu) -1 8 µm (GGH-Cu) -1 1 µm (GGH-Cu) -1 2 µm (GGH-Cu) -1 4 µm (GGH-Cu) -1 6 µm (GGH-Cu) -1 8 µm (GGH-Cu) -1 1 µm (GGH-Cu) -1 1 2 3 4 5 6 7 % Enzyme Activity 1 9 8 7 6 5 4 3 2 1 1 1 1 [I] (GGH-Cu) -1 (µm) Figure SM7 For Cu(KGHK) + 4 35 3 25 2 15 1 5 Dose Dependance Cu(KGHK) + µm (KGHK-Cu) +1 2 µm (KGHK-Cu) +1 4 µm (KGHK-Cu) +1 6 µm (KGHK-Cu) +1 8 µm (KGHK-Cu) +1 1 µm (KGHK-Cu) +1 2 µm (KGHK-Cu) +1 4 µm (KGHK-Cu) +1 6 µm (KGHK-Cu) +1 8 µm (KGHK-Cu) +1 1 µm (KGHK-Cu) +1 % Enzyme Activity 1 8 6 4 2 1 2 3 4 5 6 7 1 1 1 [I] (KGHK-Cu) +1 (µm)
This journal is The Royal Society of Chemistry 25 Figure SM8 For Cu(YIHPF) + 5 4 3 2 1 Dose Dependance Cu(YIHPF) - µm (YIHPF-Cu) -1 2 µm (YIHPF-Cu) -1 4 µm (YIHPF-Cu) -1 6 µm (YIHPF-Cu) -1 8 µm (YIHPF-Cu) -1 1 µm (YIHPF-Cu) -1 2 µm (YIHPF-Cu) -1 4 µm (YIHPF-Cu) -1 6 µm (YIHPF-Cu) -1 8 µm (YIHPF-Cu) -1 1 µm (YIHPF-Cu) -1 % Enzyme Activity 1 9 8 7 6 5 4 3 2 1 1 1 1 [I] (YIHPF-Cu) -1 (µm) 1 2 3 4 5 6 7 Figure SM9
This journal is The Royal Society of Chemistry 25 Relative Fluorescence Intensity () 6 5 4 3 2 1 E (Hydro) E (Oxd) E + Ki[I] (Hydro) E + Ki[I] (Oxd) E + 2xKi[I] 2 4 6 Time (min) Figure SM1. Plot of with time (min) under various experimental conditions (progress curve), where E(hydro) is the hydrolytic control, E(Oxd) is the oxidative control, E + K(I)(Hydro) contains the inhibitor at a concentration of 4µM under hydrolytic conditions, E + K(I)(Oxd) contains the inhibitor at a concentration of K I under oxidative conditions, and E + 2xK(I) ) contains the inhibitor at a concentration 2-fold higher than K I under oxidative conditions 3 25 E (Oxd) E + K i [I](Oxd) 2 15 1 5 5 1 15 2 25 3 Time (minutes) Figure SM11. Plot of with time (min) under oxidative experimental conditions (progress curve), where E (Oxd) is the oxidative control, E + K(I)(Oxd) contains the
This journal is The Royal Society of Chemistry 25 inhibitor at a concentration of K I under oxidative conditions.