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Michaelis-Menten Kinetics

LS Lakmali Munasinghage Silva
TS Thomas Stoll
TK Thomas Kryza
CS Carson Ryan Stephens
MH Marcus Lachlan Hastie
HI Helen Frances Irving-Rodgers
YD Ying Dong
JG Jeffrey John Gorman
JC Judith Ann Clements
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Velocity (in relative fluorescence units [RFU]) of enzyme-substrate reaction was measured with increasing substrate (MeO-Suc-Arg-Pro-Tyr-7-amino-4-methylcoumarin [AMC]) concentration (0–250 μM) using the POLARstar® Omega Plate Reader Spectrophotometer (BMG Labtech; ex 400 nm, em 505 nm; 37 °C). RFU was converted to nM using a standard curve derived using a dilution series of AMC. Kinetics values (KM, kcat and Vmax) were calculated using the non-linear regression analysis in the GraphPad Prism software. Results presented correspond to mean of RFU values +/− S.D. calculated on 6 technical replicates. D-VLR-AFC peptide substrate was used in determining KLK4 kinetics values.

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