Nzymesubstrate complex, EP is the acyl intermediate, P1 is AMC, P2 is Mu-HSSKLQ, Ks is definitely the speedy pre-equilibrium continuous (reflectingFigure two. PKA Activator list Minimum three-step PAK1 Activator manufacturer Mechanism underlying the pre steady-state and steady-state parameters for the PSA-catalyzed hydrolysis of Mu-HSSKLQ-AMC. doi:ten.1371/journal.pone.0102470.gPLOS 1 | plosone.orgEnzymatic Mechanism of PSAFigure three. Minimum reaction mechanism for the pH dependence of pre-steady-state and steady-state parameters. doi:10.1371/journal.pone.0102470.gthe actual substrate affinity for the enzyme), k2 could be the acylation rate continuous, and k3 is the deacylation rate continual [19]. Since the fluorescence spectroscopic alter is connected towards the P1 release, the enzymatic mechanism described in Figure two final results inside a biphasic kinetic pattern anytime k3,k2 [19]. Therefore, P1 release has been analyzed according to Eqn: 1 p0 : 1{e{k t zv:twhere p0 is the amplitude of the initial fast pre-steady-state phasePLOS ONE | plosone.orgEnzymatic Mechanism of PSAFigure 4. Time course of the PSA-catalyzed hydrolysis of Mu-HSSKLQ-AMC. Observation wavelength = 460 nm, pH = 7.5 and temperature = 37.0uC. The concentration of PSA was 50 nM. The concentration of Mu-HSSKLQ-AMC was 5 mM. doi:10.1371/journal.pone.0102470.g(also known as the “burst”), k is the apparent rate constant of the initial fast pre-steady-state phase, n indicates the subsequent slow steady-state process, and t is the time. The initial fast pre-steady-state kinetics (see Eqn. 1) was analyzed according to Eqns 2 and 3 [20]: p : and ‘2 k2 : S (k2 zk3 ):(Km zvkcat : : Km zwhere kcat is the catalytic constant (corresponding to the ratelimiting step), Km is the Michaelis constant, and [E] and [S] are the enzyme and substrate concentrations, respectively. Of note, the steady-state parameters kcat and Km are related to the pre-steady-state parameters Ks, k2, and k3 according to Eqns 5 and 6: kcat k2 : k3 k2 zk3 kk2 : S Ks zzkand KmThe analysis of kinetics according to Eqns. (2) and (3) allowed to determine the actual concentration of active PSA (i.e., [E]) and values of Ks, k2, and k3. The subsequent slow steady-state kinetics (see Eqn. 1) was analyzed according to Eqn. 4:Ks :k3 k2 zkPLOS ONE | plosone.orgEnzymatic Mechanism of PSAFigure 5. Dependence of k (panel A) and v (panel B) on the substrate concentration for the PSA-catalyzed hydrolysis of MuHSSKLQ-AMC. The continuous lines fitting the data reported in panels A and B were obtained according to Eqns. 3 and 4, respectively, with values of k2, k3, and Ks (panel A), and of kcat and Km (panel B) reported in Table 1. Values of pre-steady-state and steady-state parameters were obtained at pH 6.5 (o), pH 7.0 (x), pH 7.5 (+), pH 8.0 (), pH 8.5 (:), and pH 9.0 () at a temperature of 37.0uC. doi:10.1371/journal.pone.0102470.gThe pH dependence of pre-steady-state and steady-state parameters was analyzed in the framework of the minimum reaction mechanism depicted in Figure 3 [21,22], where two protonating residues are involved, according to Eqns. 7-12:obsKm 0 Km :1zKU1 : z zKU1 :KU2 : z 2 1zKL1 : z zKL1 :KL2 : z0obskcat 0 kcat :1 KL1 : z 2 KL1 :KL2 : z 2 z1 kcat : z kcat : PL PL PLobsKs 0 Ks :1zKU1 : z zKU1 :KU2 : z 2 1zKES1 : z zKES1 :KES2 : z1obsk2 0 k2 :1 KES1 : z 2 : KES1 :KES2 : z 2 z1 k2 : z k2 PES PES PESobs obsk3 0 k3 :1 KL1 : 2 : KL1 :KL2 : z1 k3 : z k3 PL PL PLzz(kcat =Km ) 0 (kcat =Km ):1 KU1 : z z1 (kcat =Km ): z PU PU(kcat =Km ):KU1 :KU2 : z 2 PU2where.