Je. Thompson et Db. Jordan, PARTITION ANALYSIS OF AN ENZYME ACTING CONCURRENTLY UPON 2 SUBSTRATESIN A CONTINUOUS MULTIWAVELENGTH ASSAY, Analytical biochemistry, 256(1), 1998, pp. 7-13
Citations number
12
Categorie Soggetti
Biology,"Biochemical Research Methods","Chemistry Analytical
We describe a multiwavelength method for measuring an enzyme's discrim
ination of one substrate over another by continuously monitoring the r
eactions of the two substrates simultaneously. This method is generall
y applicable to ultraviolet-visible diode array or rapid-scanning spec
trophotometers and the measurement requires a single incubation of enz
yme with two substrates. Rates at each of the wavelengths may be fit g
lobally by using a nonlinear least-squares fitting procedure which pro
vides adequate statistical analysis. The specificity of trypsin for N-
alpha-benzoyl-L-arginine p-nitroanilide (BRpNA) over N-t-butyloxycarbo
nyl-L-alanine-p-nitrophe nylester (BocApNP) was 2.1 as measured by the
multiwavelength partition method and 2.3 by comparing the individual
k(cat)/K-m's for the two substrates. Multiwavelength analysis was appl
ied to two enzymes in the biosynthetic pathway for fungal melanin: sey
talone dehydratase and trihydroxynaphthalene reductase from Magnaporth
ea grisea. The specificity of trihydroxynaphthalene reductase for 2,3-
dihydro-2,5-dihydroxy-4H-benzopyran-4-one compared to scytalone, a nat
ural substrate for the enzyme, was 95. Scytalone dehydratase was eight
-fold more specific for 2,3-dihydro-2,5-dihydroxy-4H-benzopyranone tha
n it was for scytalone. Multiwavelength analysis was also used to meas
ure an equilibrium constant of 0.040 for the reaction {dihydroorotate
+ oxonic acid <-> orotate + dihydrooxonic acid} catalyzed by dihydroor
otate dehydrogenase. Advantages, limitations, and further applications
of this steady-state method, which directly measures relative substra
te specificities, are delineated. All studies described in this paper
were at pH 7.0 and 25 degrees C. (C) 1998 Academic Press.