Partition analysis of an enzyme acting concurrently upon two substrates in a continuous multiwavelength assay

We describe a multiwavelength method for measuring an enzyme's discrimination of one substrate over another by continuously monitoring the reactions of the two substrates simultaneously. This method is generally applicable to ultraviolet-visible diode array or rapid-scanning spectrophotometers and the measurement requires a single incubation of enzyme with two substrates. Rates at each of the wavelengths may be fit globally by using a nonlinear least-squares fitting procedure which provides adequate statistical analysis. The specificity of trypsin for N-alpha-benzoyl-L-arginine p-nitroanilide (BRpNA) over N-t-butyloxycarbonyl-L-alanine-p-nitrophenylester (BocApNP) was 2.1 as measured by the multiwavelength partition method and 2.3 by comparing the individual kcat/K(m)'s for the two substrates. Multiwavelength analysis was applied to two enzymes in the biosynthetic pathway for fungal melanin: scytalone dehydratase and trihydroxynaphthalene reductase from Magnaporthea grisea. The specificity of trihydroxynaphthalene reductase for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one compared to scytalone, a natural substrate for the enzyme, was 95. Scytalone dehydratase was eight-fold more specific for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one than it was for scytalone. Multiwavelength analysis was also used to measure an equilibrium constant of 0.040 for the reaction ¿dihydroorotate + oxonic acid<-->orotate + dihydrooxonic acid¿ catalyzed by dihydroorotate dehydrogenase. Advantages, limitations, and further applications of this steady-state method, which directly measures relative substrate specificities, are delineated. All studies described in this paper were at pH 7.0 and 25 degrees C.