Journal: The Journal of Biological Chemistry

Article Title: Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches *

doi: 10.1074/jbc.M807510200

Figure Lengend Snippet: Kinetic schemes fitted to the experimental data. E, enzyme; S, substrate; P, product. A, simulation of N-step substrate binding mechanism. B, simulation of two-step reversible binding and one-step irreversible cleavage step mechanism used for characterization of the LF-substrate interaction. C, simulated mechanism of LF inhibition by peptide hydroxamate In-2-LF; I, inhibitor. D, mechanism of LF apoenzyme activation by Zn2+; M, metal ion (Zn2+). E, mechanism of LF apoenzyme activation by Ca2+ or Mn2+; M, metal ion (Ca2+ or Mn2+). Fitting was done with DynaFit software (15). To verify pre-steady-state kinetic data, the scree statistical test (17) was used for each mechanism (see supplemental Fig. 2 for details).

Article Snippet: The kinetic data were obtained by numerical fitting using DynaFit software (BioKin, Pullman, WA) ( 15 ), as described previously ( 16 , 17 ).

Techniques: Binding Assay, Inhibition, Activation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches *

doi: 10.1074/jbc.M807510200

Figure Lengend Snippet: Pre-steady-state kinetic parameters for LF-mediated proteolysis Pre-steady-state kinetic parameters for all proteolytic reactions except inhibition of LF15F cleavage by In-2-Lf were derived from data shown in Fig. 1 and were fitted to kinetic data ( Fig. 2 B , scheme 2 ). Inhibition of LF15F cleavage by In-2-LF was simulated as shown in Fig. 2 C , scheme 3 . All experiments were done in triplicate, and data were fitted using Dynafit software ( 15 ). K m and k cat values for pre-steady-state conditions were calculated based on equations derived from B, scheme 2 using enzyme kinetic graph theory ( 32 ). Constants for steady-state conditions were obtained using the Michaelis-Menten equation. LF15P, protein FRET substrate; LF15D, dansylated LF15 peptide; LF15F, fluorescein-labeled LF15 peptide; S20D, dansylated S20 peptide.

Article Snippet: The kinetic data were obtained by numerical fitting using DynaFit software (BioKin, Pullman, WA) ( 15 ), as described previously ( 16 , 17 ).

Techniques: Inhibition, Derivative Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches *

doi: 10.1074/jbc.M807510200

Figure Lengend Snippet: Time course for LF-mediated hydrolysis of fluorescent substrates. The reaction was monitored using a stopped-flow spectrofluorometer. Substrates used and the readout collected were as follows. A, for LF15P (LF15 peptide placed between fluorescent proteins PS-CFP2 and phiYFP), the decrease of FRET signal between fluorescent proteins was measured. B, for the fluorescein-labeled peptide substrate LF15F, the fluorescence increase was measured during LF proteolysis. C, for the dansyl-labeled peptide substrate LF15D, FRET signal between enzyme tryptophan residues and dansyl was measured. D, tryptophan-dansyl FRET was also measured in the case of the phage-selected dansyl-labeled peptide substrate S20D. Substrate concentrations used were 0.25 μm (A), 5 μm (B and C), and 4 μm (D); LF concentrations are indicated in the panels. Fig. 2B, scheme 2, was fitted to these data (smooth lines) using DynaFit software (15). Resulting parameters are given in Table 3. To build each experimental curve, data were collected in triplicate.

Article Snippet: The kinetic data were obtained by numerical fitting using DynaFit software (BioKin, Pullman, WA) ( 15 ), as described previously ( 16 , 17 ).

Techniques: Labeling, Fluorescence, Software

Journal: The Journal of Biological Chemistry

Article Title: Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches *

doi: 10.1074/jbc.M807510200

Figure Lengend Snippet: Pre-steady-state kinetics of LF15F cleavage by E687D and H690A LF mutants The pre-steady-state data were obtained by the stopped-flow method. The kinetic parameters were calculated by fitting the experimental data shown in Fig. 3 to B, scheme 2 . All kinetic curves were obtained by experiments performed in triplicate and were fitted using Dynafit kinetic software ( 15 ).

Article Snippet: The kinetic data were obtained by numerical fitting using DynaFit software (BioKin, Pullman, WA) ( 15 ), as described previously ( 16 , 17 ).

Techniques: Software

Journal: The Journal of Biological Chemistry

Article Title: Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches *

doi: 10.1074/jbc.M807510200

Figure Lengend Snippet: The pre-steady-state kinetic parameters for substrate hydrolysis by LF apoenzyme activated by divalent metal ions Pre-steady-state kinetics of substrate cleavage by LF was studied using the stopped-flow method. Prior to reaction initiation, apoenzyme and the mixture containing substrate and divalent metal ions were loaded using two separate syringes to prevent preliminary metal-enzyme and substrate-enzyme interactions. Kinetic parameters were calculated by fitting the experimental data shown in Fig. 4 to Fig. 2 D, scheme 4 , for Zn 2+ -activated apoenzyme and E, scheme 5 for Ca 2+ and Mn 2+ activation. All data were collected in triplicate and fitted using Dynafit software ( 15 ). K bindM was calculated as the sum of dissociation constants of each step of metal binding.

Article Snippet: The kinetic data were obtained by numerical fitting using DynaFit software (BioKin, Pullman, WA) ( 15 ), as described previously ( 16 , 17 ).

Techniques: Activation Assay, Software, Binding Assay