Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.
A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.
Linn
C P
CP
Becton Dickinson Research Center, Research Triangle Park, North Carolina 27709.
Mize
P D
PD
Hoke
R A
RA
Quante
J M
JM
Pitner
J B
JB
D003374:Coumarins / Q000032:analysis* / Q000138:chemical synthesis* / Q000378:metabolism; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007700:Kinetics; D015394:Molecular Structure; D012694:Serine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D014368:Tryptophanase / Q000032:analysis* / Q000378:metabolism
D003374:Coumarins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C075645:serine-7-amino-4-methylcoumarin carbamate; D012694:Serine; D014368:Tryptophanase; C028743:7-amino-4-methylcoumarin