v1
e + A = eA
v10
eAQ + B = eABQ
v11
v11
eQ_ + B = eBQ
v12
eBQ + A = eABQ
v13
eABQ = P + XQ
v14
XQ = eR
v15
eR = e + R
v16
XQ + B = XQB
v17
XQ + R = XQR
v19
e + C = eC
v2
eA + B = eAB
v20
eC + A = eAC
v21
eAC + B = eABC
v22
eC + B = eBC
v23
eBC + A = eABC
v24
eA + C = eAC
v25
eB + C = eBC
v26
eAB + C = eABC
v27
eABC = P + XC
v28
XC = eeQ
v29
eeQ = e + Q
v3
e + B = eB
v30
XC + B = XCB
v31
XC + R = XCR
v32
P = S
v4
eB + A = eAB
v5
eAB + Q = eABQ
v6
eA + Q = eAQ
v7
eB + Q = eBQ
v8
v8
e + Q = eQ_
v9
v9
eQ_ + A = eAQ
Note that constraints are not enforced in simulations. It remains the responsibility of the user to verify that simulation results satisfy these constraints.
Dissecting the catalytic mechanism of Trypanosoma brucei trypanothione synthetase by kinetic analysis and computational modeling.
J. Biol. Chem. 2013;
288
(33):
23751-23764
- PubMed: 23814051
- PubMed Central: PMC3745322
- DOI: 10.1074/jbc.M113.483289
- ISSN: 1083-351X
- URL: http://ncbi.nlm.nih.gov/pubmed/23814051
Abstract
In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl)spermidine (T(SH)2)). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays Km values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 μm, respectively, as well as Ki values for GSH and T(SH)2 of 1 mm and 360 μm, respectively. As Gsp hydrolysis has a Km value of 5.6 mm, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system's biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.
No additional notes are available for this model.