Partition Analysis of an Enzyme Acting Concurrently upon Two Substrates in a Continuous Multiwavelength Assay

Author: Thompson J.E.   Jordan D.B.  

Publisher: Elsevier

ISSN: 0003-2697

Source: Analytical Biochemistry, Vol.256, Iss.1, 1998-02, pp. : 7-13

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Abstract

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--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/Km'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 toscytalone, 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°C.

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