PARTITION ANALYSIS OF AN ENZYME ACTING CONCURRENTLY UPON 2 SUBSTRATESIN A CONTINUOUS MULTIWAVELENGTH ASSAY

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.